Prodrugs of 2,4-pyrimidinediamine compounds and their uses

ABSTRACT

The present disclosure provides prodrugs of biologically active 2,4-pyrimidinediamine compounds, salts and hydrates of the prodrugs, compositions comprising the prodrugs, intermediates and methods for synthesizing the prodrugs and methods of using the prodrugs in a variety of applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/534,784 filed Nov. 6, 2014, which is a continuation of U.S.application Ser. No. 13/861,650 filed Apr. 12, 2013, which is acontinuation of U.S. application Ser. No. 13/418,306 filed Mar. 12,2012, now U.S. Pat. No. 8,445,485, which is a continuation of U.S.application Ser. No. 11/943,506 filed Nov. 20, 2007, now U.S. Pat. No.8,163,902, which claims benefit of U.S. provisional application Ser. No.60/866,722 filed Nov. 21, 2006, the contents of each of which areincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present disclosure relates to prodrugs of biologically active2,4-pyrimidinediamine compounds, pharmaceutical compositions comprisingthe prodrugs, intermediates and synthetic methods of making the prodrugsand methods of using the prodrugs and compositions in a variety ofcontexts, such as in the treatment or prevention of various diseases.

2. Description of the Related Art

Crosslinking of Fc receptors, such as the high affinity receptor for IgE(FcεRI) and/or the high affinity receptor for IgG (FcγRI) activates asignaling cascade in mast, basophil and other immune cells that resultsin the release of chemical mediators responsible for numerous adverseevents. For example, such crosslinking leads to the release of preformedmediators of Type I (immediate) anaphylactic hypersensitivity reactions,such as histamine, from storage sites in granules via degranulation. Italso leads to the synthesis and release of other mediators, includingleukotrienes, prostaglandins and platelet-activating factors (PAFs),that play important roles in inflammatory reactions. Additionalmediators that are synthesized and released upon crosslinking Fcreceptors include cytokines and nitric oxide.

The signaling cascade(s) activated by crosslinking Fc receptors such asFcεRI and/or FcγRI comprises an array of cellular proteins. Among themost important intracellular signal propagators are the tyrosinekinases. And, an important tyrosine kinase involved in the signaltransduction pathways associated with crosslinking the FcεRI and/orFcγRI receptors, as well as other signal transduction cascades, is Sykkinase (see Valent et al., 2002, Intl. J. Hematol. 75(4):257-362 forreview).

The mediators released as a result of FcεRI and FcγRI receptorcross-linking are responsible for, or play important roles in, themanifestation of numerous adverse events. Recently, various classes of2,4-pyrimidinediamine compounds have been discovered that inhibit theFcεRI and/or FcγRI signaling cascades, and that have myriad therapeuticuses. See, e.g., U.S. application Ser. No. 10/355,543 filed Jan. 31,2003 (US 2004/0029902), international application Serial No.PCT/US03/03022 filed Jan. 31, 2003 (WO 03/063794), U.S. application Ser.No. 10/631,029 filed Jul. 29, 2003 (US 2005/0028212), internationalapplication Serial No. PCT/US03/24087 (WO 2004/014382), U.S. applicationSer. No. 10/903,263 filed Jul. 30, 2004 (US2005/0234049), andinternational application Serial No. PCT/US2004/24716 (WO 2005/016893).While many of these compounds exhibit good bioavailability properties,in some instances it may be desirable to tailor their solubility orother properties such that their bioavailability via specified routes ofadministration is optimized.

SUMMARY OF THE INVENTION

The present disclosure provides prodrugs of 2,4-pyrimidinediaminecompounds that have myriad biological activities, and hence therapeuticuses, compositions comprising the prodrugs, methods and intermediatesuseful for synthesizing the prodrugs and methods of using the prodrugsin a variety of in vitro and in vivo contexts, including in thetreatment and/or prevention of diseases mediated, at least in part, bythe activation of Fc receptor signaling cascades.

The prodrugs generally comprise a biologically active2,4-pyrimidinediamine compound that is substituted at the nitrogen atomof one or more primary or secondary amine groups with a progroup R^(p)that metabolizes or otherwise transforms under conditions of use toyield the active 2,4-pyrimidinediamine. In some embodiments, theprogroup R^(p) is a phosphorous-containing progroup. In someembodiments, the progroup includes a group or moiety that is metabolizedunder the conditions of use to yield an unstable α-hydroxymethyl,α-aminomethyl or α-thiomethyl intermediate, which then furthermetabolized in vivo to yield the active 2,4-pyrimidinediamine drug. Insome embodiments, the progroup includes an α-hydroxyalkyl, α-aminoalkylor α-thioalkyl moiety, for example an α-hydroxymethyl, α-aminomethyl,α-thiomethyl moiety, that metabolizes under the conditions of use toyield the active 2,4 pyrimidinediamine drug. For example, in someembodiments the progroup R^(p) is of the formula —CR^(d)R^(d)-AR³, whereeach R^(d) is, independently of the other, selected from hydrogen,cyano, optionally substituted (C1-C20) alkyl, (C1-C20) perfluoroalkyl,optionally substituted (C7-C30) arylalkyl and optionally substituted6-30 membered heteroarylalkyl, where each optional substituent is,independently of the others, selected from hydrogen, alkyl, aryl,arylalkyl, heteroaryl and heteroalkyl, or, alternatively, the two R^(d)are taken together with the carbon atom to which they are bonded to forma cycloalkyl containing from 3 to 8 carbon atoms; A is selected from O,S and NR⁵⁰, where R⁵⁰ is selected from hydrogen, alkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl and cycloheteroalkyl, or alternatively iscombined with R³, and, together with the nitrogen to which they areattached, form a three to seven membered ring; and R³ represents a groupthat can be metabolized in vivo to yield a group of the formula—CR^(d)R^(d)-AH, where R^(d) and A are as previously defined.

The identity of R³ is not critical, provided that it can be metabolizedunder the desired conditions of use, for example under the acidicconditions found in the stomach and/or by enzymes found in vivo, toyield a group of the formula CR^(d)R^(d)-AH, where A and R^(d) are aspreviously defined. Thus, skilled artisans will appreciate that R³ cancomprise virtually any known or later-discovered hydroxyl, amine orthiol protecting group. Non-limiting examples of suitable protectinggroups can be found, for example, in Protective Groups in OrganicSynthesis, Greene & Wuts, 2nd Ed., John Wiley & Sons, New York, 1991(especially pages 10-142 (alcohols), 277-308 (thiols) and 309-405(amines) the disclosure of which is incorporated herein by reference).

In a specific embodiment, R³ includes, together with A, an ether, athioether, a silyl ether, a silyl thioether, an ester, a thioester, anamide, a carbonate, a thiocarbonate, a carbamate, a thiocarbamate, or aurea linkage, —OCH₂SO₃R, where R is hydrogen, alkyl, aryl, arylalkyl ora metal salt (e.g., sodium, lithium, potassium); -GCH₂ ⁺N(R⁵¹)₃M⁻, whereG is absent, —OPO₃—, OSO₃— or —CO₂—, R⁵¹ is hydrogen, alkyl, aryl,arylalkyl, cycloheteroalkyl or cycloheteroalkylalkyl and M- is acounterion, usually a halide ion or the like (acetate, sulfate,phosphate, etc.). Specific exemplary embodiments include, but are notlimited to, progroups R^(p) in which R³ is selected from R^(f),—C(O)R^(f), —C(O)OR^(f), —C(O)NR^(f)R^(f) and —SiR^(f)R^(f)R^(f), whereeach R^(f) is, independently of the others, selected from hydrogen,optionally substituted lower alkyl, optionally substituted lowerheteroalkyl, optionally substituted lower cycloalkyl, optionallysubstituted lower heterocycloalkyl, optionally substituted (C6-C10)aryl, optionally substituted 5-10 membered heteroaryl, optionallysubstituted (C7-C18) arylalkyl and optionally substituted 6-18 memberedheteroarylalkyl. In a specific embodiment, each R^(f) is the same.

The identity of the progroup(s) R^(p) can be selected to tailor thewater-solubility and other properties of the underlying active2,4-pyrimidinediamine compound to be optimized for a particular mode ofadministration. It can also be selected to provide for removal atspecified organs and/or tissues within the body, such as, for example,in the digestive tract, in blood and/or serum, or via enzymes residingin specific organs, such as the liver.

In some embodiments, progroups R^(p) that are phosphorous-containingprogroups include phosphate moieties that can be cleaved in vitro byenzymes such as esterases, lipases and/or phosphatases. Such enzymes areprevalent throughout the body, residing in, for example, the stomach anddigestive tract, blood and/or serum, and in virtually all tissues andorgans. Such phosphate-containing progroups R^(p) will generallyincrease the water-solubility of the underlying active2,4-pyrimidinediamine compound, making such phosphate-containingprodrugs ideally suited for modes of administration wherewater-solubility is desirable, such as, for example, oral, buccal,intravenous, intramuscular and ocular modes of administration.

In some embodiments, each phosphate-containing progroup R^(p) in theprodrug is of the formula —(CR^(d)R^(d))_(y)—O—P(O)(OH)(OH), or a saltthereof, wherein R^(d) is as previously defined and y is an integerranging from 1 to 3, typically 1 or 2. In one specific embodiment, eachR^(d) is, independently of the others, selected from hydrogen,substituted or unsubstituted lower alkyl, substituted or unsubstitutedphenyl, substituted or unsubstituted methyl and substituted orunsubstituted benzyl. In another specific embodiment, each R^(d) is,independently of the others, selected from hydrogen and unsubstitutedlower alkyl. Specific exemplary phosphate-containing progroups R^(p)include —CH₂—O—P(O)(OH)(OH) and —CH₂CH₂—O—P(O)(OH)(OH) and/or thecorresponding salts.

While not intending to be bound by any theory of operation, when y is 1in the exemplary phosphate-containing progroups R^(p), it is believedthat the phosphate-containing prodrugs are converted in vivo by enzymessuch as phosphatases, lipases and/or esterases to the correspondinghydroxymethylamines, which are then further metabolized in vivo by theelimination of formaldehyde to yield the active 2,4-pyrimidinediaminedrug compound. The phosphate and formaldehyde metabolic by-products areinnocuous.

When y is 2 in the exemplary phosphate-containing prodrugs, it isbelieved that the prodrugs are metabolized to the active2,4-pyrimidinediamine drug compound in vivo by elimination of enolphosphate, which further metabolizes to acetaldehyde and phosphate. Thephosphate and acetaldehyde metabolic by-products are innocuous.

Skilled artisans will appreciate that certain types of precursors can beconverted in vivo to phosphate groups. Such precursors include, by wayof example and not limitation, phosphate esters, phosphites andphosphite esters. For example, phosphites can be oxidized in vivo tophosphates. Phosphate esters can be hydrolyzed in vivo to phosphates.Phosphite esters can be oxidized in vivo to phosphate esters, which canin turn be hydrolyzed in vivo to phosphates. As a consequence of theability of these phosphate precursor groups to convert to phosphates invivo, the prodrugs can also include progroups that comprise suchphosphate precursors. In some embodiments, the phosphate precursorgroups may be directly metabolized to the active 2,4-pyrimidinediaminedrug, without first being converted into a phosphate prodrug. In otherembodiments, prodrugs comprising progroups that include such phosphateprecursors are first metabolized into the corresponding phosphateprodrug, which then metabolizes to the active 2,4-pyrimidinediamine drugvia a hydroxymethylamine, as discussed above. In some embodiments, suchphosphate precursor groups are phosphate esters. The phosphate esterscan be acyclic or cyclic, and can be phosphate triesters or phosphatediesters. Such esters are generally less water-soluble than thecorresponding phosphate acid prodrugs and the corresponding active2,4-pyrimidinediamine compounds, and are therefore typically suitablefor modes of delivering prodrugs of active 2,4-pyrimidinediaminecompounds where low water-solubility is desired, including, by way ofexample and not limitation, administration via inhalation. Thesolubility of the prodrug can be specifically tailored for specificmodes of administration by appropriate selection of the number andidentity(ies) of the esterifying groups in the phosphate ester.

The mechanism by which the phosphate ester group metabolizes to thecorresponding phosphate group can be controlled by appropriate selectionof the esterifying moieties. For example, it is well-known that certainesters are acid (or base) labile, generating the corresponding phosphateunder the acidic conditions found in the stomach and digestive tract. Ininstances where it is desirable for the phosphate ester prodrug tometabolize to the corresponding phosphate prodrug in the digestive tract(such as, for example, where the prodrugs are administered orally),phosphate ester progroups that are acid-labile can be selected. Othertypes of phosphate esters are acid and base stable, being converted intothe corresponding phosphates via enzymes found in certain tissues andorgans of the body (see, e.g., the various cyclic phosphate estersdescribed in Erion et al., 2004, J. Am. Chem. Soc. 126:5154-5163,incorporated herein by reference). In instances where it is desirable toconvert a phosphate ester prodrug into the corresponding phosphateprodrug within a desired target tissue or site within the body,phosphate esters having the desired metabolic properties can beselected.

In some embodiments, each phosphate ester-containing progroup R^(p) inthe prodrug is an acyclic phosphate ester of the formula—(CR^(d)R^(d))_(y)—O—P(O)(OH)(OR^(e)) or—(CR^(d)R^(d))_(y)—O—P(O)(OR^(e))(OR^(e)), or a salt thereof, whereineach R^(e) is, independently of the others, selected from substituted orunsubstituted lower alkyl, substituted or unsubstituted (C6-C14) aryl(e.g., phenyl, naphthyl, 4-loweralkoxyphenyl, 4-methoxyphenyl),substituted or unsubstituted (C7-C20) arylalkyl (e.g., benzyl,1-phenylethan-1-yl, 2-phenylethan-1-yl), —(CR^(d)R^(d))_(y)—OR^(f),—(CR^(d)R^(d))_(y)—O—C(O)R^(f), —(CR^(d)R^(d))_(y)—O—C(O)OR^(f),—(CR^(d)R^(d))_(y)—S—C(O)R^(f), —(CR^(d)R^(d))_(y)—S—C(O)OR^(f),—(CR^(d)R^(d))_(y)—NH—C(O)R^(f), —(CR^(d)R^(d))_(y)—NH—C(O)OR^(f) and—Si(R^(d))₃, wherein R^(d), R^(f) and y are as defined above. In aspecific embodiment, each R^(d) is selected from hydrogen andunsubstituted lower alkyl and/or each R^(e) is an unsubstituted loweralkanyl or benzyl. Specific exemplary phosphate ester progroups include,but are not limited to, —CH₂—O—P(O)(OH)(OR^(e)),—CH₂CH₂—O—P(O)(OH)(OR^(e)), —CH₂—O—P(O)(OR^(e))(OR^(e)) and—CH₂CH₂—O—P(O)(OR^(e))(OR^(e)), where R^(e) is selected from loweralkanyl, i-propyl and t-butyl.

In other embodiments, each phosphate ester-containing progroup R^(p) isa cyclic phosphate ester of the formula

where each R^(g) is, independently of the others, selected from hydrogenand lower alkyl; each R^(h) is, independently of the others, selectedfrom hydrogen, substituted or unsubstituted lower alkyl, substituted orunsubstituted lower cycloheteroalkyl, substituted or unsubstituted(C6-C14) aryl, substituted or unsubstituted (C7-C20) arylalkyl andsubstituted or unsubstituted 5-14 membered heteroaryl; z is an integerranging from 0 to 2; and R^(d) and y are as previously defined. In aspecific embodiment, each phosphate ester-containing progroup R^(p) is acyclic phosphate ester of the formula

where R^(d), R^(h) and y are as previously defined.

The mechanism by which cyclic phosphate ester prodrugs including suchcyclic phosphate ester progroups metabolize in vivo to the active drugcompound depends, in part, on the identity of the R^(h) substitutent.For example, cyclic phosphate ester progroups in which each R^(h) is,independently of the others, selected from hydrogen and lower alkyl arecleaved in vivo by esterases. Thus, in some embodiments, the cyclicphosphate ester progroups are selected such that they are cleavable invivo by esterases. Specific examples of such cyclic phosphate esterprogroups include, but are not limited to, progroups selected from

Alternatively, cyclic phosphate ester prodrugs having progroups in whichthe R^(h) substituents are substituted or unsubstituted aryl, arylalkyland heteroaryl groups, are not typically cleaved by esterases, but areinstead metabolized to the active prodrug by enzymes, such as cytochromeP₄₅₀ enzymes, that reside in the liver. For example, a series of cyclicphosphate ester nucleotide prodrugs that undergo an oxidative cleavagereaction catalyzed by a cytochrome P₄₅₀ enzyme (CYP) expressedpredominantly in the liver are described in Erion et al., 2004, J. Am.Chem. Soc. 126:5154-5163. In some embodiments, the cyclic phosphateester progroups are selected such that they are cleavable by CYP enzymesexpressed in the liver. Specific exemplary embodiments of such cyclicphosphate ester-containing progroups R^(p) include, but are not limitedto, progroups having the formula

where R^(h) is selected from phenyl, 3-chlorophenyl, 4-pyridyl and4-methoxyphenyl.

As skilled artisans will appreciate, phosphites and phosphite esters canundergo oxidation in vivo to yield the corresponding phosphate andphosphate ester analogs. Such reactions can be carried out in vivo by,for example, oxidase enzymes, oxoreductase enzymes and other oxidativeenzymes. Thus, the phosphorous-containing progroups R^(p) can alsoinclude phosphite and phosphite ester analogs of any of the phosphateand phosphate ester progroups described above. In some embodiments thephosphorous-containing progroups R^(p) include, but are not limited to,groups of the formula —(CR^(d)R^(d))_(y)—O—P(OH)(OH),—(CR^(d)R^(d))_(y)—O—P(OH)(OR^(e)) and—(CR^(d)R^(d))_(y)—O—P(OR^(e))(R^(e)), or salts thereof, where R^(d),R^(e) and y are as previously defined. Specific exemplary embodimentsinclude groups in which each R^(d) is, independently of the others,selected from hydrogen and unsubstituted lower alkyl and/or each R^(e)is, independently of the others, selected from unsubstituted loweralkanyl and benzyl. Specific exemplary acyclic phosphite andphosphite-ester progroups include, but are not limited to,—CH₂—O—P(OH)(OH), —CH₂CH₂—O—P(OH)(OH), —CH₂—O—P(OH)(OR^(e)), and—CH₂CH₂—O—P(OR^(e))(OR^(e)), where each R^(e) is selected from loweralkanyl, i-propyl and t-butyl. Specific exemplary cyclic phosphite esterprodrugs include phosphite analogs of the above-described cyclicphosphate ester progroups. Conceptually, prodrug compounds includingsuch phosphite and/or phosphite ester progroups can be thought of asprodrugs of the corresponding phosphate and phosphate ester prodrugs.

As mentioned above, it is believed that certain phosphate-containingprodrugs metabolize in vivo through the correspondinghydroxymethylamines. Although these hydroxymethylamines metabolize invivo to the corresponding active 2,4-pyrimidinediamine compounds, theyare stable at pH 7 and can be prepared and administered ashydroxyalkyl-containing prodrugs. In some embodiments, eachhydroxyalkyl-containing progroup R^(p) of such prodrugs is of theformula —CR^(d)R^(d)—OH, where R^(d) is as previously defined. Aspecific exemplary hydroxyalkyl-containing progroup R^(p) is —CH₂OH.

Virtually any known 2,4-pyrimidinediamine compound that has biological,and hence therapeutic, activity can be protected at an available primaryor secondary amine with one or more progroups R^(p) as described herein.Suitable active 2,4-pyrimidinediamine compounds are described, forexample, in U.S. application Ser. No. 10/355,543 filed Jan. 31, 2003(US2004/0029902A1), international application Serial No. PCT/US03/03022filed Jan. 31, 2003 (WO 03/063794), U.S. application Ser. No. 10/631,029filed Jul. 29, 2003 (US 2005/0028212), international application SerialNo. PCT/US03/24087 (WO2004/014382), U.S. application Ser. No. 10/903,263filed Jul. 30, 2004 (US2005/0234049), and international applicationSerial No. PCT/US2004/24716 (WO 2005/016893), the disclosures of whichare incorporated herein by reference. In such 2,4-pyrimidinediaminecompounds, the progroup(s) R^(p) can be attached to any availableprimary or secondary amine, including, for example, the N2 nitrogen atomof the 2,4-pyrimidinediamine moiety, the N4 nitrogen atom of the2,4-pyrimidinediamine moiety, and/or a primary or secondary nitrogenatom included in a substituent on the 2,4-pyrimidinediamine compound.The use of phosphate-containing progroups R^(p) is especially useful for2,4-pyrimidinediamine compounds that exhibit poor water solubility underphysiological conditions (for example, solubilities of less than about10 μg/ml). While not intending to be bound by any theory of operation,it is believed that the phosphate-containing progroups aid thesolubility of the underlying active 2,4-pyrimidinediamine compound,which in turn increases its bioavailability when administered orally. Itis believed that the phosphate progroups R^(p) are metabolized byphosphatase enzymes found in the digestive tract, permitting uptake ofthe underlying active drug.

It has been discovered that the water solubility and oralbioavailability of a particular biologically active2,4-pyrimidinediamine compound, illustrated below (Compound 1),increased dramatically when formulated to include a progroup R^(p) ofthe formula —CH₂—O—P(O)(OH)₂ at the ring nitrogen atom highlighted withthe asterisk (Compound 4):

Significantly, whereas the water solubility of the active drug(Compound 1) is in the range of about <1 μg/ml in aqueous buffer underphysiological conditions, the solubility of the corresponding phosphateprodrug (Compound 4) is greater than 5 mg/ml under the same conditions,or approximately 2000 times greater. This increased water-solubilityallows for better dissolution in the gut, thereby facilitating oraladministration. Other active 2,4-pyrimidinediamine compounds havingsimilarly poor water solubilities are expected to exhibit similarincreases in water solubility and oral bioavailability when formulatedas phosphate prodrugs.

As mentioned above, phosphate ester prodrugs are generally lesswater-soluble than the corresponding phosphate prodrugs, and aretherefore generally useful in applications where low water-solubility isdesired, such as, for example, administration via inhalation. The sameholds true for the relative water-solubility of phosphite ester andphosphite prodrugs.

In some embodiments, the prodrugs described herein are2,4-pyrimidinediamine compounds that are substituted at the N4 nitrogenof the 2,4-pyrimidinediamine moiety with a substituted or unsubstitutednitrogen-containing bicyclic ring that includes at least one progroupR^(p) as described herein at one or more of: the nitrogen atom(s) of thebicyclic ring, the N2 nitrogen of the 2,4-pyrimidinediamine moietyand/or the N4 nitrogen of the 2,4-pyrimidinediamine moiety. In aspecific illustrative exemplary embodiment, the prodrug is a compoundaccording to structural formula (I):

including salts, solvates, hydrates and N-oxides thereof, wherein:

Y is selected from CH₂, NR²⁴, O, S, S(O) and S(O)₂;

Z¹ and Z² are each, independently of one another, selected from CH andN;

R² is an optionally substituted lower alkyl, lower cycloalkyl, lowerheteroalkyl, lower cycloheteroalkyl, aryl, phenyl, or heteroaryl group;

R⁵ is an electronegative group, such as, for example, a halo, fluoro,cyano, nitro, trihalomethyl or trifluoromethyl group;

R¹⁷ is selected from hydrogen, halogen, fluoro, lower alkyl and methylor, alternatively, R¹⁷ may be taken together with R¹⁸ to form an oxo(═O) group or, together with the carbon atom to which they are attached,a spirocycle containing from 3 to 7 carbon atoms;

R¹⁸ is selected from hydrogen, halogen, fluoro, lower alkyl and methylor, alternatively, R¹⁸ may be taken together with R¹⁷ to form an oxo(═O) group or, together with the carbon atom to which they are attached,a spirocycle containing from 3 to 7 carbon atoms;

R¹⁹ is selected from hydrogen, lower alkyl, and methyl or,alternatively, R¹⁹ may be taken together with R²⁰ to form an oxo (═O)group or, together with the carbon atom to which they are attached, aspirocycle containing from 3 to 7 carbon atoms;

R²⁰ is selected from hydrogen, lower alkyl and methyl or, alternatively,R²⁰ may be taken together with R¹⁹ to form an oxo (═O) group or,together with the carbon atom to which they are attached, a spirocyclecontaining from 3 to 7 carbon atoms;

R²¹, R²² and R²³ are each, independently of one another, selected fromhydrogen and a progroup R^(p) as described herein; and

R²⁴ is selected from hydrogen, lower alkyl and a progroup R^(p) asdescribed herein, with the proviso that at least one of R²¹, R²², R²³and R²⁴ must be a progroup R^(p). In some embodiments, each of R²¹, R²²and R²³ is one of the specific progroups exemplified above and R²⁴ ishydrogen. In some embodiments R²¹ is one of the specific progroupsexemplified above and R²², R²³ and R²⁴ are each hydrogen. In someembodiments, R²¹, R²² and R²³ are each one of the specific progroupsexemplified above and R²⁴ is lower alkyl.

In another aspect, the present disclosure provides compositionscomprising one or more of the prodrugs described herein and anappropriate carrier, excipient or diluent. The exact nature of thecarrier, excipient or diluent will depend upon the desired use for thecomposition, and may range from being suitable or acceptable forveterinary uses to being suitable or acceptable for human use. Thecomposition may optionally include one or more additional compounds.

In still another aspect, the present disclosure provides intermediatesuseful for synthesizing the prodrugs described herein. In the case ofphosphate- or phosphite-containing prodrugs, the intermediates generallycomprise prodrugs in which the oxygen atoms of the phosphate- and/orphosphite-containing progroups are masked with protecting groups thatare selectively removable under specified conditions. In someembodiments, the protecting groups are selectively removable undermildly acidic conditions. In some embodiments, the intermediates arephosphate or phosphite esters which are themselves prodrugs that can bemetabolized into active 2,4-pyrimidinediamine compounds. In oneillustrative embodiment, the intermediates include prodrugs in whicheach R^(p) progroup is, independently of the others, of the formula—(CR^(d)R^(d))_(y)—O—P(O)(OR^(i))(OR^(i)),—(CR^(d)R^(d))y-O—P(O)(OR^(i))(OH), —(CR^(d)R^(d))y-O—P(OR^(i))(OR^(i))or —(CR^(d)R^(d))y-O—P(OR^(i))(OH), where each R^(i) is, independentlyof the others, selected from lower unsubstituted alkanyl, substituted orunsubstituted phenyl and substituted or unsubstituted benzyl, and R^(d)and y are as previously defined. In a specific embodiment, theintermediates include phosphate and/or phosphite esters in which eachR^(i) is, independently of the others, selected from lower linearalkanyl, lower branched alkanyl, i-propyl, t-butyl and lower cyclicalkanyl.

In some embodiments, the intermediates comprise an active2,4-pyrimidinediamine that is substituted at a nitrogen atom of aprimary or secondary amine group with a group of the formula—CR^(d)R^(d)-AH, where R^(d) and A are as previously defined.

Another aspect of the invention relates to salts of the above-describedprodrug compounds. For example, in one aspect of the invention theprodrug is a prodrug salt, or a hydrate, solvate or N-oxide thereof,comprising a 2,4-pyrimidinediamine moiety and at least one progroup saltR^(q) linked covalently to a primary or secondary amino nitrogen atom ofthe 2,4-pyrimidinediamine moiety. The 2,4-pyrimidinediamine moiety maybe, for example, any of the 2,4-pyrimidinediamine moieties describedabove. The progroup salt R^(q) may be any suitable salt of any acidicprogroup R^(p). For example, the progroup salt R^(q) may be a salt of aphosphate progroup, a carbonate progroup, or a sulfonate progroup. Thecounterion may be, for example, an alkali cation, an alkaline earthcation or an ammonium cation. Examples of suitable progroup salts R^(q)include: —(CR^(d)R^(d))_(y)—O—P(O)(O⁻)²M⁺ ₂,—(CR^(d)R^(d))_(y)—O—P(O)(OR^(e))(O⁻)M⁺, —(CR^(d)R^(d))_(y)—O—P(O⁻)₂M⁺₂, —(CR^(d)R^(d))_(y)—O—P(OR^(e))(O⁻)M⁺, and—(CR^(d)R^(d))_(y)—O—COO⁻M⁺, wherein R^(d), R^(e), and y are asdescribed above and M⁺ is as described below.

As used herein, the term “salt” refers not only to species comprised ofanions and cations, but also to other forms in which the bonds amongatoms are not entirely ionic, e.g., coordination complexes. Similarly,for convenience, chemical structures may be drawn as combinations ofanions and cations, e.g., A⁻B⁺, but this is not intended to imply thatthe bonds are necessarily purely ionic.

Another aspect of the invention relates to methods for producingcrystalline forms of the salts of the above-described prodrug compounds.In one embodiment of this aspect, the crystalline form of the salt ofthe produgs described herein may contain solvent in the crystal. In apreferred embodiment, the solvent in the crystalline form of the salt ofthe prodrug is one or more molecules of water per molecule of prodrug.

In yet another aspect, the present disclosure provides methods ofsynthesizing the intermediates and/or prodrugs described herein.Phosphate-containing prodrugs can be synthesized by reacting an active2,4-pyrimidinediamine compound with a phosphate ester halide, forexample, a phosphate ester halide of the formulaX—(CR^(d)R^(d))_(y)—O—P(O)(OR^(j))(OR^(j)) orX—(CR^(d)R^(d))_(y)—O—P(O)(OR^(j))(OH), where each is, independently ofthe others, a selectively removable protecting group; X is a halide,such as, for example, chloride; and R^(d) and y are as previouslydefined. In some embodiments, each R^(j) is R^(e), where as previouslydefined. Removal of the selectively removable protecting groups R^(j)yields a phosphate prodrug. In some embodiments each R^(j) is the sameand is selected from lower linear alkyl, lower branched alkyl and lowercycloalkyl. In some embodiments, each R^(j) is isopropyl or t-butyl. Inembodiments in which mixtures of intermediates are obtained, forexample, mixtures of intermediates which contain different numbers ofprogroups or progroups at different positions on the2,4-pyrimidinediamine molecule, the desired intermediate can be isolatedfrom the mixture using standard separation and/or isolation techniques(e.g., column chromatography). Alternatively, a desired prodrug can beisolated from a mixture of different prodrugs using standard separationand/or isolation techniques.

Acyclic phosphate ester prodrugs can be obtained in an analogous mannerby reacting the active 2,4-pyrimidinediamine with a phosphate esterhalide, for example a phosphate ester halide of the formulaX—(CR^(d)R^(d))_(y)—O—P(O)(OH)(OR^(e)) orX—(CR^(d)R^(d))_(y)—O—P(O)(OR^(e))(OR^(e)), where X, R^(d), y and R^(e)are as previously defined. In this instance, removal of the esterifyinggroups R^(e) is not necessary.

Acyclic phosphite and phosphite ester prodrugs can be prepared in ananalogous manner from the corresponding phosphite ester halides, forexample phosphite ester halides of the formulaX—(CR^(d)R^(d))_(y)—O—P(OR^(j))(OR^(j)),X—(CR^(d)R^(d))_(y)—O—P(OR^(e))(OH),X—(CR^(d)R^(d))_(y)—O—P(OR^(e))(OR^(e)), where X, R^(d), y, R^(e) andR^(j) are as previously defined.

Cyclic phosphate ester and phosphite ester prodrugs can be prepared byreacting the active 2,4-pyrimidinediamine compound with thecorresponding cyclic phosphate ester or phosphite ester halide, forexample, a cyclic phosphate ester halide of the formula

or a cyclic phosphite ester halide of the formula

where X, R^(d), y, z, R^(g) and R^(h) are as previously defined.

Embodiments in which R^(p) is —CR^(d)R^(d)-AR³ can be prepared from thecorresponding 2,4-pyrimidinediamine drug using conventional methods. Forexample, when A is O, the intermediates can be synthesized by reactingan active 2,4-pyrimidinediamine compound, with an aldehyde or ketone ofthe formula R^(d)—C(O)—R^(d), where R^(d) is as previously defined, toyield a corresponding hydroxymethylamine intermediate (where R^(p) is—CR^(d)R^(d)—OH). The hydroxymethylamine intermediate can then beconverted into the prodrug using standard techniques. In accordance withthe definition of R^(p), the hydroxymethylamine intermediate is also aprodrug of the invention. For example, other drug substances containingsecondary amines have been added to formaldehyde to afford theircorresponding isolable hydroxymethylamine adducts, Bansal et al., J.Pharmaceutical Sci. 1981, 70: (8), 850-854; Bansal et al., J.Pharmaceutical Sci. 1981, 70: (8), 855-856; Khan et al., J.Pharmaceutical and Biomedical Analysis 1989, 7 (6), 685-691.Alternatively, hydroxyalkyl-containing prodrugs can be prepared in twosteps by first reacting the active 2,4-pyrimidinediamine with abis-functional electrophile, such as a halide of the formulaX¹—CR^(d)R^(d)—X², where X¹ represents a first halide, X² represents asecond halide and R^(d) is as previously defined. In a specificexemplary embodiment, the halide is of the formula I—CR^(d)R^(d)—Cl. Theunreacted halide is then hydroxylated to yield thehydroxyalkyl-containing prodrug using standard techniques.

Prodrugs in which A is O, S or NR⁵⁰ can be synthesized fromcorresponding N-methyl phosphate esters. According to this embodiment,the phosphate ester groups can be displaced with a group of the formulaR³-AH, where R³ and A are as previously defined, to yield the prodrug,as discussed in further detail below.

Prodrug salts can be prepared by treating an appropriate conjugate acidprodrug compound with a base, performing any desired cationreplacements, and isolating the desired prodrug salt through standardtechniques such as precipitation or concentration, or according to themethods described herein.

Crystalline forms of prodrug salts can be prepared according to methodsknown in the art, or according to the methods described herein. Suchcrystalline forms may contain only the prodrug salt in the crystal, ormay contain a second chemical species in the crystal. For example,crystal forms of the prodrug salts of the present invention may containone or more solvent molecules per prodrug salt molecule, i.e. a solvate.Examples of solvents which may be contained in the crystalline forms ofthe prodrugs of the instant invention are water, methanol, ethanol,isopropanol, acetone, and the like. A preferred embodiment of theinvention is a hydrate of the prodrug salts described herein.

Many of the prodrugs described herein, and in particular the prodrugsaccording to structural formula (I), metabolize to yield2,4-pyrimidinediamine compounds that are potent inhibitors ofdegranulation of immune cells, such as mast, basophil, neutrophil and/oreosinophil cells. Additional 2,4-pyrimidinediamine compounds that exertsimilar biological activities that can be formulated as prodrugs asdescribed herein and used in the various methods described herein aredescribed in U.S. application Ser. No. 10/355,543 filed Jan. 31, 2003(US2004/0029902A1), international application Serial No. PCT/US03/03022filed Jan. 31, 2003 (WO 03/063794), U.S. application Ser. No. 10/631,029filed Jul. 29, 2003 (US 2005/0028212), international application SerialNo. PCT/US03/24087 (WO2004/014382), U.S. application Ser. No. 10/903,263filed Jul. 30, 2004 (US2005/0234049), and international applicationSerial No. PCT/US2004/24716 (WO 2005/016893), the disclosures of whichare incorporated herein by reference. Thus, in still another aspect, thepresent disclosure provides methods of regulating, and in particularinhibiting, degranulation of such cells. The method generally involvescontacting a cell that degranulates with an amount of a suitable prodrugdescribed herein, or an acceptable salt, hydrate, solvate, N-oxideand/or composition thereof, effective to regulate or inhibitdegranulation of the cell. The method may be practiced in in vitrocontexts provided that the contacting is performed under conditions inwhich the progroup(s) metabolize to yield the active2,4-pyrimidinediamine compound, or in in vivo contexts as a therapeuticapproach towards the treatment or prevention of diseases characterizedby, caused by or associated with cellular degranulation.

While not intending to be bound by any theory of operation, biochemicaldata confirm that many of these active 2,4-pyrimidinediamine compoundsexert their degranulation inhibitory effect, at least in part, byblocking or inhibiting the signal transduction cascade(s) initiated bycrosslinking of the high affinity Fc receptors for IgE (“FcεRI”) and/orIgG (“FcγRI”) (see, e.g., U.S. application Ser. No. 10/631,029 filedJul. 29, 2003 (US 2005/0028212), international application Serial No.PCT/US03/24087 (WO2004/014382), U.S. application Ser. No. 10/903,263filed Jul. 30, 2004 (US2005/0234049), and international applicationSerial No. PCT/US2004/24716 (WO 2005/016893), the disclosures of whichare incorporated herein by reference. Indeed, these active2,4-pyrimidinediamine compounds are potent inhibitors of bothFcεRI-mediated and FcγRI-mediated degranulation. As a consequence, theprodrugs described herein may be used to inhibit these Fc receptorsignaling cascades in any cell type expressing such FcεRI and/or FcγRIreceptors including but not limited to macrophages, mast, basophil,neutrophil and/or eosinophil cells.

The methods also permit the regulation of, and in particular theinhibition of, downstream processes that result as a consequence ofactivating such Fc receptor signaling cascade(s). Such downstreamprocesses include, but are not limited to, FcεRI-mediated and/orFcγRI-mediated degranulation, cytokine production and/or the productionand/or release of lipid mediators such as leukotrienes andprostaglandins. The method generally involves contacting a cellexpressing an Fc receptor, such as one of the cell types discussedabove, with an amount of a prodrug described herein, or an acceptablesalt, hydrate, solvent, N-oxide and/or composition thereof, effective toregulate or inhibit the Fc receptor signaling cascade and/or adownstream process effected by the activation of this signaling cascade.The method may be practiced in in vitro contexts provided that thecontacting is performed under conditions under which the progroup(s)metabolize to yield the active 2,4-pyrimidinediamine compound, or in invivo contexts as a therapeutic approach towards the treatment orprevention of diseases characterized by, caused by or associated withthe Fc receptor signaling cascade, such as diseases effected by therelease of granule specific chemical mediators upon degranulation, therelease and/or synthesis of cytokines and/or the release and/orsynthesis of lipid mediators such as leukotrienes and prostaglandins.

In yet another aspect, the present disclosure provides methods oftreating and/or preventing diseases characterized by, caused by orassociated with the release of chemical mediators as a consequence ofactivating Fc receptor signaling cascades, such as FcεRI and/orFcγRI-signaling cascades. The methods may be practiced in animals inveterinary contexts or in humans. The methods generally involveadministering to an animal subject or a human an amount of a prodrugdescribed herein, or an acceptable salt, hydrate, solvate, N-oxideand/or composition thereof, effective to treat or prevent the disease.As discussed previously, activation of the FcεRI or FcγRI receptorsignaling cascade in certain immune cells leads to the release and/orsynthesis of a variety of chemical substances that are pharmacologicalmediators of a wide variety of diseases. Any of these diseases may betreated or prevented according to the methods of the invention.

For example, in mast cells and basophil cells, activation of the FcεRIor FcγRI signaling cascade leads to the immediate (i.e., within 1-3 min.of receptor activation) release of preformed mediators of atopic and/orType I hypersensitivity reactions (e.g., histamine, proteases such astryptase, etc.) via the degranulation process. Such atopic or Type Ihypersensitivity reactions include, but are not limited to, anaphylacticreactions to environmental and other allergens (e.g., pollens, insectand/or animal venoms, foods, drugs, contrast dyes, etc.), anaphylactoidreactions, hay fever, allergic conjunctivitis, allergic rhinitis,allergic asthma, atopic dermatitis, eczema, urticaria, mucosaldisorders, tissue disorders and certain gastrointestinal disorders.

The immediate release of the preformed mediators via degranulation isfollowed by the release and/or synthesis of a variety of other chemicalmediators, including, among other things, platelet activating factor(PAF), prostaglandins and leukotrienes (e.g., LTC4) and the de novosynthesis and release of cytokines such as TNFα, IL-4, IL-5, IL-6,IL-13, etc. The first of these two processes occurs approximately 3-30min. following receptor activation; the latter approximately 30 min.-7hrs. following receptor activation. These “late stage” mediators arethought to be in part responsible for the chronic symptoms of theabove-listed atopic and Type I hypersensitivity reactions, and inaddition are chemical mediators of inflammation and inflammatorydiseases (e.g., osteoarthritis, inflammatory bowel disease, ulcerativecolitis, Crohn's disease, idiopathic inflammatory bowel disease,irritable bowel syndrome, spastic colon, etc.), low grade scarring(e.g., scleroderma, increased fibrosis, keloids, post-surgical scars,pulmonary fibrosis, vascular spasms, migraine, reperfusion injury andpost myocardial infarction), and sicca complex or syndrome. All of thesediseases may be treated or prevented according to the methods describedherein.

Additional diseases that can be treated or prevented according to themethods described herein include diseases associated with basophil celland/or mast cell pathology. Examples of such diseases include, but arenot limited to, diseases of the skin such as scleroderma, cardiacdiseases such as post myocardial infarction, pulmonary diseases such aspulmonary muscle changes or remodeling and chronic obstructive pulmonarydisease (COPD), diseases of the gut such as inflammatory bowel syndrome(spastic colon), acute myeloid leukemia (AML) and immunethrombocytopenic purpura.

Many of the active 2,4-pyrimidinediamine compounds are also potentinhibitors of the tyrosine kinase Syk kinase. Examples of such2,4-pyrimidinediamine are described, for example, in U.S. applicationSer. No. 10/355,543 filed Jan. 31, 2003 (US2004/0029902A1),international application Serial No. PCT/US03/03022 filed Jan. 31, 2003(WO 03/063794), U.S. application Ser. No. 10/631,029 filed Jul. 29, 2003(US 2005/0028212), international application Serial No. PCT/US03/24087(WO2004/014382), U.S. application Ser. No. 10/903,263 filed Jul. 30,2004 (US2005/0234049), and international application Serial No.PCT/US2004/24716 (WO 2005/016893), the disclosures of which areincorporated herein by reference. Thus, in still another aspect, thepresent disclosure provides methods of regulating, and in particularinhibiting, Syk kinase activity. The method generally involvescontacting a Syk kinase or a cell comprising a Syk kinase with an amountof a suitable prodrug, or an acceptable salt, hydrate, solvate, N-oxideand/or composition thereof, effective to regulate or inhibit Syk kinaseactivity. In one embodiment, the Syk kinase is an isolated orrecombinant Syk kinase. In another embodiment, the Syk kinase is anendogenous or recombinant Syk kinase expressed by a cell, for example amast cell or a basophil cell. The method may be practiced in in vitrocontexts provided that the contacting is performed under conditionsunder which the progroup(s) metabolize to yield the active2,4-pyrimidinediamine compound, or in in vivo contexts as a therapeuticapproach towards the treatment or prevention of diseases characterizedby, caused by or associated with Syk kinase activity.

While not intending to be bound by any particular theory of operation,it is believed that such active 2,4-pyrimdinediamine compounds inhibitcellular degranulation and/or the release of other chemical mediatorsprimarily by inhibiting Syk kinase that gets activated through the gammachain homodimer of FcεRI. This gamma chain homodimer is shared by otherFc receptors, including FcγRI, FcγRIII and FcαRI. For all of thesereceptors, intracellular signal transduction is mediated by the commongamma chain homodimer. Binding and aggregation of those receptorsresults in the recruitment and activation of tyrosine kinases such asSyk kinase. As a consequence of these common signaling activities, theprodrugs described herein that metabolize to such active2,4-pyrimidinediamine compounds may be used to regulate, and inparticular inhibit, the signaling cascades of Fc receptors having thisgamma chain homodimer, such as FcεRI, FcγRI, FcγRIII and FcαRI, as wellas the cellular responses elicited through these receptors.

Syk kinase is known to play a critical role in other signaling cascades.For example, Syk kinase is an effector of B-cell receptor (BCR)signaling (Turner et al., 2000, Immunology Today 21:148-154) and is anessential component of integrin beta(1), beta(2) and beta(3) signalingin neutrophils (Mocsai et al., 2002, Immunity 16:547-558). Active2,4-pyrimidinediamine compounds that are potent inhibitors of Syk kinasecan be used to regulate, and in particular inhibit, any signalingcascade where Syk plays a role, such as, for example, the Fc receptor,BCR and integrin signaling cascades, as well as the cellular responseselicited through these signaling cascades. Thus, the prodrugs describedherein that metabolize to such active 2,4-pyrimidinediamine compoundscan be used to regulate such activities. The particular cellularresponse regulated or inhibited will depend, in part, on the specificcell type and receptor signaling cascade, as is well known in the art.Non-limiting examples of cellular responses that may be regulated orinhibited with such prodrugs include a respiratory burst, cellularadhesion, cellular degranulation, cell spreading, cell migration,phagocytosis (e.g., in macrophages), calcium ion flux (e.g., in mast,basophil, neutrophil, eosinophil and B-cells), platelet aggregation, andcell maturation (e.g., in B-cells).

Thus, in another aspect, the present disclosure provides methods ofregulating, and in particular inhibiting, signal transduction cascadesin which Syk plays a role. The method generally involves contacting aSyk-dependent receptor or a cell expressing a Syk-dependent receptorwith an amount of a suitable prodrug described herein, or an acceptablesalt, hydrate, solvate, N-oxide and/or composition thereof, effective toregulate or inhibit the signal transduction cascade. The methods mayalso be used to regulate, and in particular inhibit, downstreamprocesses or cellular responses elicited by activation of the particularSyk-dependent signal transduction cascade. The methods may be practicedto regulate any signal transduction cascade where Syk is now known orlater discovered to play a role. The methods may be practiced in invitro contexts provided that the contacting is performed underconditions under which the progroup(s) metabolize to yield the active2,4-pyrimidinediamine compound, or in in vivo contexts as a therapeuticapproach towards the treatment or prevention of diseases characterizedby, caused by or associated with activation of the Syk-dependent signaltransduction cascade. Non-limited examples of such diseases includethose previously discussed.

Recent studies have shown that activation of platelets by collagen ismediated through the same pathway used by immune receptors, with animmunoreceptor tyronsine kinase motif on the FcRγ playing a pivotal role(Watson & Gibbons, 1998, Immunol. Today 19:260-264), and also that FcRγplays a pivotal role in the generation of neointimal hyperplasiafollowing balloon injury in mice, most likely through collagen-inducedactivation of platelets and leukocyte recruitment (Konishi et al., 2002,Circulation 105:912-916). Thus, the prodrugs described herein can alsobe used to inhibit collagen-induced platelet activation and to treat orprevent diseases associated with or caused by such platelet activation,such as, for example, intimal hyperplasia and restenosis followingvascular injury.

Cellular and animal data also confirm that many of these active2,4-pyrimidinediamine compounds may also be used to treat or preventautoimmune diseases and/or symptoms of such diseases (see, e.g., U.S.application Ser. No. 10/631,029 filed Jul. 29, 2003 (US 2005/0028212),international application Serial No. PCT/US03/24087 (WO2004/014382),U.S. application Ser. No. 10/903,263 filed Jul. 30, 2004(US2005/0234049), and international application Serial No.PCT/US2004/24716 (WO 2005/016893), the disclosures of which areincorporated herein by reference. As a consequence, prodrugs of suchactive 2,4-pyrimidinediamine compounds can likewise be used to treat orprevent such autoimmune diseases and/or symptoms. The methods generallyinvolve administering to a subject suffering from an autoimmune diseaseor at risk of developing an autoimmune disease an amount of a suitableprodrug described herein, or an acceptable salt, N-oxide, hydrate,solvate or composition thereof, effective to treat or prevent theautoimmune disease and/or its associated symptoms. Autoimmune diseasesthat can be treated or prevented with the prodrugs include thosediseases that are commonly associated with nonanaphylactichypersensitivity reactions (Type II, Type III and/or Type IVhypersensitivity reactions) and/or those diseases that are mediated, atleast in part, by activation of the FcγR signaling cascade in monocytecells. Such autoimmune disease include, but are not limited to, thoseautoimmune diseases that are frequently designated as single organ orsingle cell-type autoimmune disorders and those autoimmune disease thatare frequently designated as involving systemic autoimmune disorder.Non-limiting examples of diseases frequently designated as single organor single cell-type autoimmune disorders include: Hashimoto'sthyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritisof pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis,Goodpasture's disease, autoimmune thrombocytopenia, sympatheticophthalmia, myasthenia gravis, Graves' disease, primary biliarycirrhosis, chronic aggressive hepatitis, ulcerative colitis andmembranous glomerulopathy. Non-limiting examples of diseases oftendesignated as involving systemic autoimmune disorder include: systemiclupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter'ssyndrome, polymyositis-dermatomyositis, systemic sclerosis,polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.Additional autoimmune diseases, which can be β-cell (humoral) based orT-cell based, include autoimmune alopecia, Type I or juvenile onsetdiabetes, and thyroiditis.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B provide schemes illustrating metabolic pathways ofexemplary phosphorous-containing prodrugs;

FIG. 2 provides a scheme illustrating a metabolic pathway of anexemplary cyclic phosphate ester prodrug;

FIG. 3 illustrates an exemplary synthesis of exemplary cyclic phosphateprodrug;

FIGS. 4-11 provide graphs illustrating various pharmacokinetic data fordrug Compound 1 and/or prodrug Compound 4; and

FIGS. 12-15 provide graphs illustrating various structural data forprodrug salt hydrate 32.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following terms are intended to have the followingmeanings:

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C1-C6 means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyl s such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-di en-1-yl, buta-1,3-di en-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-di en-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. As used herein,“lower alkyl” means (C1-C8) alkyl.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like. Asused herein, “lower alkanyl” means (C1-C8) alkanyl.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). Typical alkenylgroups include, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-di en-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like. As used herein, “lower alkenyl” means (C2-C8) alkenyl.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. As used herein, “loweralkynyl” means (C2-C8) alkynyl.

“Alkyldiyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon group having the stated number of carbon atoms (i.e., C1-C6means from one to six carbon atoms) derived by the removal of onehydrogen atom from each of two different carbon atoms of a parentalkane, alkene or alkyne, or by the removal of two hydrogen atoms from asingle carbon atom of a parent alkane, alkene or alkyne. The twomonovalent radical centers or each valency of the divalent radicalcenter can form bonds with the same or different atoms. Typicalalkyldiyl groups include, but are not limited to, methandiyl; ethyldiylssuch as ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-di en-1,1-diyl, buta-1,3-dien-1,2-diyl, buta-1,3-di en-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Whereit is specifically intended that the two valencies are on the samecarbon atom, the nomenclature “alkylidene” is used. In some embodiments,the alkyldiyl group is (C1-C8) alkyldiyl. Specific embodiments includesaturated acyclic alkanyldiyl groups in which the radical centers are atthe terminal carbons, e.g., methandiyl (methano); ethan-1,2-diyl(ethano); propan-1,3-diyl (propano); butan-1,4-diyl (butano); and thelike (also referred to as alkylenos, defined infra).

“Alkyleno” by itself or as part of another substituent refers to astraight-chain saturated or unsaturated alkyldiyl group having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. The locant of a doublebond or triple bond, if present, in a particular alkyleno is indicatedin square brackets. Typical alkyleno groups include, but are not limitedto, methano; ethylenos such as ethano, etheno, ethyno; propylenos suchas propano, prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.; butylenossuch as butano, but[1]eno, but[2]eno, buta[1,3]dieno, but[1]yno,but[2]yno, buta[1,3]diyno, etc.; and the like. Where specific levels ofsaturation are intended, the nomenclature alkano, alkeno and/or alkynois used. In some embodiments, the alkyleno group is (C1-C8) or (C1-C3)alkyleno. Specific embodiments include straight-chain saturated alkanogroups, e.g., methano, ethano, propano, butano, and the like.

“Heteroalkyl,” Heteroalkanyl,” Heteroalkenyl,” Heteroalkynyl,”Heteroalkyldiyl” and “Heteroalkyleno” by themselves or as part ofanother substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyland alkyleno groups, respectively, in which one or more of the carbonatoms are each independently replaced with the same or differentheteratoms or heteroatomic groups. Typical heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—, —S(O)NR′—, —S(O)₂NR′—, and the like, including combinations thereof, whereeach R′ is independently hydrogen or (C1-C8) alkyl.

“Cycloalkyl” and “Heterocycloalkyl” by themselves or as part of anothersubstituent refer to cyclic versions of “alkyl” and “heteroalkyl”groups, respectively. For heteroalkyl groups, a heteroatom can occupythe position that is attached to the remainder of the molecule. Typicalcycloalkyl groups include, but are not limited to, cyclopropyl;cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such ascyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl andcyclohexenyl; and the like. Typical heterocycloalkyl groups include, butare not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl,piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl,morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl,piperazin-2-yl, etc.), and the like.

“Acyclic Heteroatomic Bridge” refers to a divalent bridge in which thebackbone atoms are exclusively heteroatoms and/or heteroatomic groups.Typical acyclic heteroatomic bridges include, but are not limited to,—O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—, —S(O) NR′—, —S(O)₂NR′—,and the like, including combinations thereof, where each R′ isindependently hydrogen or (C1-C8) alkyl.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene,tetrahydronaphthalene, etc. Typical parent aromatic ring systemsinclude, but are not limited to, aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and thelike.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C6-C15 means from 6 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene, and the like, as well as the various hydroisomers thereof. In preferred embodiments, the aryl group is (C6-C15)aryl, with (C6-C10) being more typical. Specific exemplary aryls includephenyl and naphthyl.

“Arylaryl” by itself or as part of another substituent refers to amonovalent hydrocarbon group derived by the removal of one hydrogen atomfrom a single carbon atom of a ring system in which two or moreidentical or non-identical parent aromatic ring systems are joineddirectly together by a single bond, where the number of such direct ringjunctions is one less than the number of parent aromatic ring systemsinvolved. Typical arylaryl groups include, but are not limited to,biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, andthe like. Where the number of carbon atoms in an arylaryl group arespecified, the numbers refer to the carbon atoms comprising each parentaromatic ring. For example, (C6-C15) arylaryl is an arylaryl group inwhich each aromatic ring comprises from 6 to 15 carbons, e.g., biphenyl,triphenyl, binaphthyl, phenylnaphthyl, etc. In some embodiments, eachparent aromatic ring system of an arylaryl group is independently a(C6-C15) aromatic, more preferably a (C6-C10) aromatic. Specificexemplary arylaryl groups include those in which all of the parentaromatic ring systems are identical, e.g., biphenyl, triphenyl,binaphthyl, trinaphthyl, etc.

“Biaryl” by itself or as part of another substituent refers to anarylaryl group having two identical parent aromatic systems joineddirectly together by a single bond. Typical biaryl groups include, butare not limited to, biphenyl, binaphthyl, bianthracyl, and the like. Insome embodiments, the aromatic ring systems are (C6-C15) aromatic rings,more typically (C6-C10) aromatic rings. A particular exemplary biarylgroup is biphenyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or spa carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylakenyl and/orarylalkynyl is used. In some embodiments, the arylalkyl group is(C7-C21) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C1-C6) and the aryl moiety is (C6-C15). In somespecific embodiments the arylalkyl group is (C7-C13), e.g., the alkanyl,alkenyl or alkynyl moiety of the arylalkyl group is (C1-C3) and the arylmoiety is (C6-C10).

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms are each independently replacedwith the same or different heteroatoms or heteroatomic groups. Typicalheteroatoms or heteroatomic groups to replace the carbon atoms include,but are not limited to, N, NH, P, O, S, S(O), S(O)₂, Si, etc.Specifically included within the definition of “parent heteroaromaticring systems” are fused ring systems in which one or more of the ringsare aromatic and one or more of the rings are saturated or unsaturated,such as, for example, benzodioxan, benzofuran, chromane, chromene,indole, indoline, xanthene, etc. Also included in the definition of“parent heteroaromatic ring system” are those recognized rings thatinclude common substituents, such as, for example, benzopyrone and1-methyl-1,2,3,4-tetrazole. Specifically excluded from the definition of“parent heteroaromatic ring system” are benzene rings fused to cyclicpolyalkylene glycols such as cyclic polyethylene glycols. Typical parentheteroaromatic ring systems include, but are not limited to, acridine,benzimidazole, benzisoxazole, benzodioxan, benzodioxole, benzofuran,benzopyrone, benzothiadiazole, benzothiazole, benzotriazole,benzoxaxine, benzoxazole, benzoxazoline, carbazole, β-carboline,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic group having the stated number of ring atoms(e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof. In preferredembodiments, the heteroaryl group is a 5-14 membered heteroaryl, with5-10 membered heteroaryl being particularly preferred.

“Heteroaryl-Heteroaryl” by itself or as part of another substituentrefers to a monovalent heteroaromatic group derived by the removal ofone hydrogen atom from a single atom of a ring system in which two ormore identical or non-identical parent heteroaromatic ring systems arejoined directly together by a single bond, where the number of suchdirect ring junctions is one less than the number of parentheteroaromatic ring systems involved. Typical heteroaryl-heteroarylgroups include, but are not limited to, bipyridyl, tripyridyl,pyridylpurinyl, bipurinyl, etc. Where the number of atoms are specified,the numbers refer to the number of atoms comprising each parentheteroaromatic ring systems. For example, 5-15 memberedheteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which eachparent heteroaromatic ring system comprises from 5 to 15 atoms, e.g.,bipyridyl, tripuridyl, etc. In some embodiments, each parentheteroaromatic ring system is independently a 5-15 memberedheteroaromatic, more typically a 5-10 membered heteroaromatic. Specificexemplary heteroaryl-heteroaryl groups include those in which all of theparent heteroaromatic ring systems are identical.

“Biheteroaryl” by itself or as part of another substituent refers to aheteroaryl-heteroaryl group having two identical parent heteroaromaticring systems joined directly together by a single bond. Typicalbiheteroaryl groups include, but are not limited to, bipyridyl,bipurinyl, biquinolinyl, and the like. In some embodiments, theheteroaromatic ring systems are 5-15 membered heteroaromatic rings, moretypically 5-10 membered heteroaromatic rings.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or spa carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylakenyl and/orheteroarylalkynyl is used. In some embodiments, the heteroarylalkylgroup is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is (C1-C6) alkyl and theheteroaryl moiety is a 5-15-membered heteroaryl. In some specificexemplary embodiments, the heteroarylalkyl is a 6-13 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C1-C3)alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.

“Halogen” or “Halo” by themselves or as part of another substituent,unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen atoms is replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C1-C2) haloalkyl” includes fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

The above-defined groups may include prefixes and/or suffixes that arecommonly used in the art to create additional well-recognizedsubstituent groups. As examples, “alkyloxy” or “alkoxy” refers to agroup of the formula —OR″, “alkylamine” refers to a group of the formula—NHR″ and “dialkylamine” refers to a group of the formula —NR″R″, whereeach R″ is independently an alkyl. As another example, “haloalkoxy” or“haloalkyloxy” refers to a group of the formula —OR′″, where R″′ is ahaloalkyl.

“Substituted,” when used to modify a specified group or radical, meansthat one or more hydrogen atoms of the specified group or radical areeach, independently of one another, replaced with the same or differentsubstituent(s). Substituent groups useful for substituting for hydrogenson saturated carbon atoms in the specified group or radical include, butare not limited to —R⁶⁰, halo, —O⁻M⁺, ═O, —OR⁷⁰, ═S, —NR⁸⁰R⁸⁰, —NR⁷⁰,═N—OR⁷⁰, trihalomethyl, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃,—S(O)₂R⁷⁰, —S(O)₂O⁻ M⁺, —S(O)₂OR⁷⁰, —OS(O)₂R⁷⁰, —OS(O)₂O⁻M⁺,—OS(O)₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂, —P(O)(OR⁷⁰)O⁻ M⁺, —P(O)(OR⁷⁰)(OR⁷⁰),—C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —C(O)O⁻ M⁺, —C(O)OR⁷⁰, —C(S)OR⁷⁰,—C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰—OC(O)O⁻ M⁺,—OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰C(O)O⁻ M⁺,—NR⁷⁰C(O)OR⁷⁰, —NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and—NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰ is selected from the group consisting ofalkyl, cycloalkyl, heteroalkyl, cycloheteroalkyl, aryl, arylalkyl,heteroaryl and heteroarylalkyl; each R⁷⁰ is independently hydrogen orR⁶⁰; each R⁸⁰ is independently R⁷⁰ or alternatively, the two R⁸⁰'s,taken together with the nitrogen atom to which they are bonded, form a5-, 6- or 7-membered cycloheteroalkyl which may optionally include from1 to 4 of the same or different additional heteroatoms selected from thegroup consisting of O, N and S; and each M⁺ is a counter ion with asingle positive charge. Each M+ may independently be, for example, analkali ion, such as K⁺, Na⁺, Li⁺; an ammonium ion, such as ⁺N(R⁶⁰)₄; oran alkaline earth ion, such as [Ca²⁺]_(0.5), [Mg²⁺]_(0.5), or[Ba²⁺]_(0.5). As specific examples, —NR⁸⁰R⁸⁰ is meant to include —NH₂,—NH-alkyl, N-pyrrolidinyl and N-morpholinyl.

Similarly, substituent groups useful for substituting for hydrogens onunsaturated carbon atoms in the specified group or radical include, butare not limited to, —R⁶⁰, halo, —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸⁰,trihalomethyl, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)₂R⁷⁰,—S(O)₂O⁻M⁺, —S(O)₂OR⁷⁰, —OS(O)₂R⁷⁰, —OS(O)₂O⁻M⁺, —OS(O)₂OR⁷⁰,—P(O)(O⁻)₂(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)(OR⁷⁰), —C(O)R⁷⁰, —C(S)R⁷⁰,—C(NR⁷⁰)R⁷⁰, —C(O)O⁻M⁺, —C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰,—C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰, —OC(O)O⁻M⁺, —OC(O)OR⁷⁰,—OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰C(O)O⁻M⁺, —NR⁷⁰C(O)OR⁷⁰,—NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and—NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ are as previouslydefined.

Substituent groups, other than R^(p), useful for substituting forhydrogens on nitrogen atoms in heteroalkyl and cycloheteroalkyl groupsinclude, but are not limited to, —R⁶⁰, —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, —S⁻M⁺,—NR⁸⁰R⁸⁰, trihalomethyl, —CF₃, —CN, —NO, —NO₂, —S(O)₂R⁷⁰, —S(O)₂O⁻M⁺,—S(O)₂OR⁷⁰, —OS(O)₂R⁷⁰, —OS(O)₂O⁻M⁺, —OS(O)₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂,—P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰ (OR⁷⁰), —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰,—C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰,—OC(S)R⁷⁰, —OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰,—NR⁷⁰C(O)OR⁷⁰, —NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and—NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ are as previouslydefined.

Substituent groups from the above lists useful for substituting othergroups or atoms specified as “substituted” will be apparent to those ofskill in the art.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene and Wuts,Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, John Wiley &Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative aminoprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxyl protecting groupsinclude, but are not limited to, those where the hydroxyl group iseither acylated or alkylated such as benzyl and trityl ethers, as wellas alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g.,TMS or TIPPS groups) and allyl ethers.

“Fc Receptor” refers to a member of the family of cell surface moleculesthat binds the Fc portion (containing the specific constant region) ofan immunoglobulin. Each Fc receptor binds immunoglobulins of a specifictype. For example the Fcα receptor (“FcαR”) binds IgA, the FcεR bindsIgE and the FcγR binds IgG.

The FcαR family includes the polymeric Ig receptor involved inepithelial transport of IgA/IgM, the myeloid specific receptor RcαRI(also called CD89), the Fcα/μR and at least two alternative IgAreceptors (for a recent review see Monteiro & van de Winkel, 2003, Annu.Rev. Immunol, advanced e-publication). The FcγRI is expressed onneutrophils, eosinophils, monocytes/macrophages, dendritic cells andkupfer cells. The FcαRI includes one alpha chain and the FcR gammahomodimer that bears an activation motif (ITAM) in the cytoplasmicdomain and phosphorylates Syk kinase.

The FcεR family includes two types, designated FcεRI and FcεRII (alsoknown as CD23). FcεRI is a high affinity receptor (binds IgE with anaffinity of about 10¹⁰M⁻¹) found on mast, basophil and eosinophil cellsthat anchors monomeric IgE to the cell surface. The FcεRI possesses onealpha chain, one beta chain and the gamma chain homodimer discussedabove. The FcεRII is a low affinity receptor expressed on mononuclearphagocytes, B lymphocytes, eosinophils and platelets. The FcεRIIcomprises a single polypeptide chain and does not include the gammachain homodimer.

The FcγR family includes three types, designated FcγRI (also known asCD64), FcγRII (also known as CD32) and FcγRIII (also known as CD16).FcγRI is a high affinity receptor (binds IgG1 with an affinity of10⁸M⁻¹) found on mast, basophil, mononuclear, neutrophil, eosinophil,dendritic and phagocyte cells that anchors nomomeric IgG to the cellsurface. The FcγRI includes one alpha chain and the gamma chain dimershared by FcαRI and FcεRI.

The FcγRII is a low affinity receptor expressed on neutrophils,monocytes, eosinophils, platelets and B lymphocytes. The FcγRII includesone alpha chain, and does not include the gamma chain homodimerdiscussed above.

The FcγRIII is a low affinity (binds IgG1 with an affinity of 5×10⁵M⁻¹)expressed on NK, eosinophil, macrophage, neutrophil and mast cells. Itcomprises one alpha chain and the gamma homodimer shared by FcαRI, FcεRIand FcγRI.

Skilled artisans will recognize that the subunit structure and bindingproperties of these various Fc receptors, as well as the cell typesexpressing them, are not completely characterized. The above discussionmerely reflects the current state-of-the-art regarding these receptors(see, e.g., Immunobiology: The Immune System in Health & Disease, 5^(th)Edition, Janeway et al., Eds, 2001, ISBN 0-8153-3642-x, FIG. 9.30 at pp.371), and is not intended to be limiting with respect to the myriadreceptor signaling cascades that can be regulated with the prodrugsdescribed herein.

“Fc Receptor-Mediated Degranulation” or “Fc Receptor-InducedDegranulation” refers to degranulation that proceeds via an Fc receptorsignal transduction cascade initiated by crosslinking of an Fc receptor.

“IgE-Induced Degranulation” or “FcεRI-Mediated Degranulation” refers todegranulation that proceeds via the IgE receptor signal transductioncascade initiated by crosslinking of FcεR1-bound IgE. The crosslinkingmay be induced by an IgE-specific allergen or other multivalent bindingagent, such as an anti-IgE antibody. In mast and/or basophil cells, theFcεRI signaling cascade leading to degranulation may be broken into twostages: upstream and downstream. The upstream stage includes all of theprocesses that occur prior to calcium ion mobilization. The downstreamstage includes calcium ion mobilization and all processes downstreamthereof. Compounds that inhibit FcεRI-mediated degranulation may act atany point along the FcεRI-mediated signal transduction cascade.Compounds that selectively inhibit upstream FcεRI-mediated degranulationact to inhibit that portion of the FcεRI signaling cascade upstream ofthe point at which calcium ion mobilization is induced. In cell-basedassays, compounds that selectively inhibit upstream FcεRI-mediateddegranulation inhibit degranulation of cells such as mast or basophilcells that are activated or stimulated with an IgE-specific allergen orbinding agent (such as an anti-IgE antibody) but do not appreciablyinhibit degranulation of cells that are activated or stimulated withdegranulating agents that bypass the FcεRI signaling pathway, such as,for example the calcium ionophores ionomycin and A23187.

“IgG-Induced Degranulation” or “FcγRI-Mediated Degranulation” refers todegranulation that proceeds via the FcγRI signal transduction cascadeinitiated by crosslinking of FcγRI-bound IgG. The crosslinking may beinduced by an IgG-specific allergen or another multivalent bindingagent, such as an anti-IgG or fragment antibody. Like the FcεRIsignaling cascade, in mast and basophil cells the FcγRI signalingcascade also leads to degranulation which may be broken into the sametwo stages: upstream and downstream. Similar to FcγRI-mediateddegranulation, compounds that selectively inhibit upstreamFcγRI-mediated degranulation act upstream of the point at which calciumion mobilization is induced. In cell-based assays, compounds thatselectively inhibit upstream FcγRI-mediated degranulation inhibitdegranulation of cells such as mast or basophil cells that are activatedor stimulated with an IgG-specific allergen or binding agent (such as ananti-IgG antibody or fragment) but do not appreciably inhibitdegranulation of cells that are activated or stimulated withdegranulating agents that bypass the FcγRI signaling pathway, such as,for example the calcium ionophores ionomycin and A23187.

“Ionophore-Induced Degranulation” or “Ionophore-Mediated Degranulation”refers to degranulation of a cell, such as a mast or basophil cell, thatoccurs upon exposure to a calcium ionophore such as, for example,ionomycin or A23187.

“Syk Kinase” refers to the well-known 72 kDa non-receptor (cytoplasmic)spleen protein tyrosine kinase expressed in B-cells and otherhematopoetic cells. Syk kinase includes two consensus Src-homology 2(SH2) domains in tandem that bind to phosphorylated immunoreceptortyrosine-based activation motifs (“ITAMs”), a “linker” domain and acatalytic domain (for a review of the structure and function of Sykkinase see Sada et al., 2001, J. Biochem. (Tokyo) 130:177-186); see alsoTurner et al., 2000, Immunology Today 21:148-154). Syk kinase has beenextensively studied as an effector of B-cell receptor (BCR) signaling(Turner et al., 2000, supra). Syk kinase is also critical for tyrosinephosphorylation of multiple proteins which regulate important pathwaysleading from immunoreceptors, such as Ca²⁺ mobilization andmitogen-activated protein kinase (MAPK) cascades and degranulation. Sykkinase also plays a critical role in integrin signaling in neutrophils(see, e.g., Mocsai et al. 2002, Immunity 16:547-558).

As used herein, Syk kinase includes kinases from any species of animal,including but not limited to, homosapiens, simian, bovine, porcine,rodent, etc., recognized as belonging to the Syk family. Specificallyincluded are isoforms, splice variants, allelic variants, mutants, bothnaturally occurring and man-made. The amino acid sequences of such Sykkinases are well known and available from GENBANK. Specific examples ofmRNAs encoding different isoforms of human Syk kinase can be found atGENBANK accession no. gi|21361552|ref|NM_003177.2|,gi|496899|emb|Z29630.1|HSSYKPTK[496899] andgi|15030258|gb|BC011399.1|BC011399[15030258], which are incorporatedherein by reference.

Skilled artisans will appreciate that tyrosine kinases belonging toother families may have active sites or binding pockets that are similarin three-dimensional structure to that of Syk. As a consequence of thisstructural similarity, such kinases, referred to herein as “Syk mimics,”are expected to catalyze phosphorylation of substrates phosphorylated bySyk. Thus, it will be appreciated that such Syk mimics, signaltransduction cascades in which such Syk mimics play a role, andbiological responses effected by such Syk mimics and Syk mimic-dependentsignaling cascades may be regulated, and in particular inhibited, withmany of the prodrugs described herein.

“Syk-Dependent Signaling Cascade” refers to a signal transductioncascade in which Syk kinase plays a role. Non-limiting examples of suchSyk-dependent signaling cascades include the FcαRI, FcεRI, FcγRI,FcγRIII, BCR and integrin signaling cascades.

“Autoimmune Disease” refers to those diseases which are commonlyassociated with the nonanaphylactic hypersensitivity reactions (Type II,Type III and/or Type IV hypersensitivity reactions) that generallyresult as a consequence of the subject's own humoral and/orcell-mediated immune response to one or more immunogenic substances ofendogenous and/or exogenous origin. Such autoimmune diseases aredistinguished from diseases associated with the anaphylactic (Type I orIgE-mediated) hypersensitivity reactions.

The Prodrug Compounds

As described in the Summary, the instant disclosure provides prodrugs ofbiologically active 2,4-pyrimidinediamine compounds, such as the various2,4-pyrimidinediamine compounds described in U.S. application Ser. No.10/355,543 filed Jan. 31, 2003 (US2004/0029902A1), internationalapplication Serial No. PCT/US03/03022 filed Jan. 31, 2003 (WO03/063794), U.S. application Ser. No. 10/631,029 filed Jul. 29, 20032005/0028212), international application Serial No. PCT/US03/24087(WO2004/014382), U.S. application Ser. No. 10/903,263 filed Jul. 30,2004 (US2005/0234049), and international application Serial No.PCT/US2004/24716 (WO 2005/016893), the disclosures of which areincorporated herein by reference. Prodrugs of these2,4-pyrimidinediamine compounds are of particular interest, as thesecompounds inhibit upstream Fc receptor signaling cascades as well as Sykkinase and Syk kinase-dependent signaling cascades. The prodrugsgenerally include such active 2,4-pyrimidinediamine compounds in whichone or more of the available primary or secondary amine groups is maskedwith a progroup R^(p) that metabolizes in vivo to yield the active 2,4pyrimidinediamine drug. As also discussed in the Summary section, and aswill be discussed in more detail, below, the nature of the progroup canvary, and will depend upon, among other factors, the desired watersolubility of the prodrug, its intended mode of administration and/orits intended mechanism or site of metabolism to the active2,4-pyrimidinediamine compound.

For example, it has been discovered that a specific active2,4-pyrimidinediamine drug (Compound 1, below), exhibits vastly superiorwater solubility when formulated as a phosphate prodrug (Compound 4,below):

Compound Structure Solubility Compound 1

<1 μg/ml Compound 4

>5 mg/ml

This prodrug Compound 4 also exhibits superior bioavailability comparedto the corresponding active drug Compound 1 when administered orally totest animals. In fact, unlike the drug Compound 1, absorption of theprodrug Compound 4 is not dependent upon formulation. Inpharmacokinetics studies carried out in rats, the prodrug Compound 4 wasabsorbed equally well from solutions (e.g., PEG-400 solutions andcarboxymethylcellulose solutions) and powders (packed in hard gelatincapsules). While not intending to be bound by any particular theory ofoperation, it is believed that the improved oral bioavailability of theprodrug Compound 4, as well as its formulation-independent absorption,is due, at least in part, to its higher water-solubility. It is expectedthat other active 2,4-pyrimidinediamine compounds that have similarlylow water solubilities, and hence oral bioavailabilities, will exhibitsimilar increases in water solubility and oral bioavailability whenformulated as phosphate prodrugs. As will be described more fully below,it may be desirable to formulate the active 2,4-pyrimidinediaminecompounds as phosphate salts in order to provide high solubility andstability.

Conversely, the corresponding phosphate ester prodrug of active drugCompound 1 would be expected to have lower water-solubility than theactive Compound 1 compound. Thus, it is expected that phosphate esterprodrugs of active 2,4-pyrimidinediamine compounds that have lowerwater-solubility than the corresponding active 2,4-pyrimidinediaminecompounds will be especially useful in applications and formulationswhere low water-solubility is desirable, such as formulations adaptedfor delivery via inhalation.

One class of active 2,4-pyrimidinediamine compounds that is expected tobenefit from formulation as prodrugs, and in particular as phosphateprodrugs, includes 2,4-pyrimidinediamines in which the N4-substituent ofthe 2,4-pyrimidinediamine moiety is a substituted or unsubstitutednitrogen-containing heteroaryl ring of the formula

where Z¹ and Z² are each, independently of one another, selected from CHand N and Y is selected from CH₂, NH, O, S, S(O) and S(O)₂. Suchprodrugs can include progroups R^(p) at: one or both of the non-aromaticring nitrogens of the heteroaryl ring, the N2-nitrogen of the2,4-pyrimidinedimaine moiety, the N4-nitrogen atom of the2,4-pyrimidinediamine moiety and/or any available nitrogen atoms in thesubstituent attached to the N2 nitrogen atom of the2,4-pyrimidinediamine moiety.

In one illustrative embodiment, the prodrugs are compounds according tostructural formula (I):

including salts, solvates, hydrates and N-oxides thereof, wherein:

Y is selected from CH₂, NR²⁴, O, S, S(O) and S(O)₂;

Z¹ and Z² are each, independently of one another, selected from CH andN;

R² is selected from lower alkyl optionally substituted with one or moreof the same or different R⁸ groups, lower cycloalkyl optionallysubstituted with one or more of the same or different R⁸ groups,cyclohexyl optionally substituted with one or more of the same ordifferent R⁸ groups, 3-8 membered cycloheteroalkyl optionallysubstituted with one or more of the same or different R⁸ groups,(C6-C14) aryl optionally substituted with one or more of the same ordifferent R⁸ groups, phenyl optionally substituted with one or more ofthe same or different R⁸ groups and 5-15 membered heteroaryl optionallysubstituted with one or more of the same or different R⁸ groups;

R⁵ is selected from halo, fluoro, cyano, nitro, trihalomethyl andtrifluoromethyl;

R⁸ is selected from R^(a), R^(b), R^(a) substituted with one or more,for example, from one to four, of the same or different R^(a) or R^(b),—OR^(a) substituted with one or more of the same or different R^(a) orR^(b), —B(OR^(a))₂, —B(NR^(c)R^(c))₂, —(CH₂)_(m)—R^(b),—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—R^(b), —S—(CH₂)_(m)—R^(b),—O—CHR^(a)R^(b), —O—CR^(a)(R^(b))₂, —O—(CHR^(a))_(m)—R^(b),—O—(CH₂)_(m)—CH[(CH₂)_(m)R^(b)]R^(b), —S—(CHR^(a))_(m)—R^(b),—C(O)NH—(CH₂)_(m)—R^(b), —C(O)NH—(CHR^(a))_(m)—R^(b),—O—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b),—S—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b),—O—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b),—S—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), —NH—(CH₂)_(m)—R^(b),—NH—(CHR^(a))_(m)—R^(b), —NH[(CH₂)_(m)R^(b)], —N[(CH₂)_(m)R^(b)]₂,—NH—C(O)—NH—(CH₂)_(m)—R^(b), —NH—C(O)—(CH₂)_(m)—CHR^(b)R^(b) and—NH—(CH₂)_(m)—C(O)—NH—(CH₂)_(m)—R^(b);

R¹⁷ is selected from hydrogen, halogen, fluoro, lower alkyl and methylor, alternatively, R¹⁷ may be taken together with R¹⁸ to form an oxo(═O) group or, together with the carbon atom to which they are attached,a spirocycle containing from 3 to 7 carbon atoms;

R¹⁸ is selected from hydrogen, halogen, fluoro, lower alkyl and methylor, alternatively, R¹⁸ may be taken together with R¹⁷ to form an oxo(═O) group or, together with the carbon atom to which they are attached,a spirocycle containing from 3 to 7 carbon atoms;

R¹⁹ is selected from hydrogen, lower alkyl, and methyl or,alternatively, R¹⁹ may be taken together with R²⁰ to form an oxo (═O)group or, together with the carbon atom to which they are attached, aspirocycle containing from 3 to 7 carbon atoms;

R²⁰ is selected from hydrogen, lower alkyl and methyl or, alternatively,R²⁰ may be taken together with R¹⁹ to form an oxo (═O) group or,together with the carbon atom to which they are attached, a spirocyclecontaining from 3 to 7 carbon atoms;

each R^(a) is, independently of the others, selected from hydrogen,lower alkyl, lower cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl,(C6-C10) aryl, phenyl, (C7-C16) arylalkyl, benzyl, 2-6 memberedheteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl, piperazinyl,homopiperazinyl, piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10membered heteroaryl and 6-16 membered heteroarylalkyl;

each R^(b) is a suitable group independently selected from ═O, —OR′,(C1-C3) haloalkyloxy, ═S, —SR^(a), ═NR^(a), ═NOR^(a), —NR^(c)R^(c),

halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂,

═N₂, —N₃, —S(O)₂R^(a), —S(O)₂R^(a), —S(O)₂OR^(a), —S(O)NR^(c)R^(c),—S(O)₂NR^(c)R^(c), —OS(O)R^(a), —OS(O)₂R^(a), —OS(O)₂OR^(a),—OS(O)₂NR^(c)R^(c), —C(O)R^(a), —C(O)OR^(a), —C(O)NR^(c)R^(c),—C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a),—C(NOH)NR^(c)R^(c), —OC(O)R^(a), —OC(O)OR^(a), —OC(O)NR^(c)R^(c),—OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c), —[NHC(O)]_(n)R^(a),—[NR^(a)C(O)]_(n)R^(a), —[NHC(O)]_(n)OR^(a), —[NR^(a)C(O)]_(n)OR^(a),—[NHC(O)]_(n)NR^(c)R^(c), —[NR^(a)C(O)]_(n)NR^(c)R^(c),—[NHC(NH)]_(n)NR^(c)R^(c) and —[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c);

each R^(c) is, independently of the others, selected from a protectinggroup and R^(a), or, alternatively, the two R^(c) bonded to the samenitrogen atom are taken together with that nitrogen atom to form a 5 to8-membered cycloheteroalkyl or heteroaryl which may optionally includeone or more of the same or different additional heteroatoms and whichmay optionally be substituted with one or more, for example, from one tofour, of the same or different R^(a) groups;

R²¹, R²² and R²³ are each, independently of one another, selected fromhydrogen and a progroup R^(p);

R²⁴ is selected from hydrogen, lower alkyl and progroup R^(p);

each m is, independently of the others, an integer from 1 to 3; and

each n is, independently of the others, an integer from 0 to 3, with theproviso that at least one of R²¹, R²², R²³ and R²⁴ is a progroup.

In the prodrugs described herein, and in particular in the prodrugs ofstructural formula (I), R²¹, R²² and R²³ each represent either hydrogenor a progroup R^(p). Also, R²⁴ represents hydrogen, a lower alkyl or aprogroup R^(p). Thus, the prodrugs can include a single R′ progroup, twoR^(p) progroups, three R^(p) progroups, or even more R^(p) progroups,depending, in part, on the identity of Y and whether the R² substituentincludes any R^(p) progroups. In some embodiments, it is preferred thatthe prodrugs described herein, and in particular the prodrugs ofstructural formula (I), include only one R^(p) group. Without intendingto be bound by any theory of operation, it is possible that thedifferent R^(p) groups in prodrugs including more than one R^(p)progroup may metabolize at different rates. Prodrugs including a singleR^(p) progroup would avoid such differential metabolic kinetics. Aspecific embodiment of prodrugs according to structural formula (I) thatinclude a single progroup R^(p) are compounds according to structuralformula (Ia):

wherein Y¹ is selected from CH₂, NR²⁴, O, S, S(O) and S(O)₂; and Z², R²,R⁵, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²⁴ and R^(p) are as previously defined, withthe proviso that R² does not include any R^(p) groups.

The identity of any R^(p) progroups present in the prodrugs describedherein is not critical for success, provided that it hydrolyzes underthe conditions of use to yield the active 2,4-pyrimidinediaminecompound. As will be described more fully below, in certain embodimentsof the invention, the R^(p) progroup is a progroup salt R^(q).

It has recently been discovered that a phosphate-containing prodrugaccording to the structure illustrated below:

metabolizes in vivo to the corresponding active 2,4-pyrimidinediaminecompound (Compound 1), illustrated below:

While not intending to be bound by any particular theory of operation,it is believed that this prodrug metabolizes to active Compound 1 viathe corresponding hydroxymethylamine intermediate illustrated below:

Such hydroxymethylamine compound are known to be unstable underphysiological conditions and various pH ranges where they hydrolyze invivo to yield formaldehyde and the active drug substance. Based on thisobservation, it is believed that prodrugs that include hydroxyl“protecting” groups that can be metabolized in vivo, for example by theacidic conditions of the stomach and/or by enzymes present in thedigestive tract or other organs and/or tissues or fluids with the body,to yield the hydroxymethylamine intermediate illustrated above willlikewise metabolize to the active 2,4 pyrimidinediamine drug.

Moreover, it is expected that the amino and thio analogs of thishydroxymethylamine intermediate, will be similarly unstable atphysiological conditions and also hydrolyze in vivo to the active2,4-pyrimdiendiamine drug. Accordingly, it is also expected that thecorresponding amino and thio compounds, as well as compounds in whichthe α-amino and α-thio groups are masked with “protecting” groups thatare removed under physiological conditions of use to yield the α-aminoand α-thio groups, will likewise make suitable prodrugs.

Thus, in some embodiments, the progroup(s) R′ in the prodrugs ofstructural formulae (I) and (Ia) are of the formula CR^(d)R^(d)-A-R³,where each R^(d) is, independently of the other, selected from hydrogen,cyano, —C(O)R^(e), —C(O)OR^(e), —C(O)NR^(e)R^(e), —C(OR^(e))(OR^(e)),optionally substituted (C1-C20) alkyl, (C1-C20) perfluoroalkyl,optionally substituted (C7-C30) arylalkyl and optionally substituted6-30 membered heteroarylalkyl, where each R^(e) is, independently of theothers, selected from hydrogen, alkyl (for example lower alkyl), aryl(for example phenyl or naphthyl, arylalkyl (for example benzyl),heteroaryl and heteroarylalkyl; A is selected from O, S and NR⁵⁰, whereR⁵⁰ is selected from R^(d) and cycloalkyl, or, alternatively, is takentogether with R³ such that R⁵⁰ and R³, together with nitrogen atom towhich they are attached, form a three-to seven-membered ring; and R³ isa group that, together with A, metabolizes under the conditions of useto yield an intermediate group of the formula —CR^(d)R^(d)AH, whereR^(d) and A are as previously defined. As mentioned above, compounds ofstructural formula (I) and (Ia) in which the R^(p) groups are of theformula —CR^(d)R^(d)-AH spontaneously hydrolyze in vivo to yield theactive 2,4-pyrimidinediamine drug.

The mechanism by which the R³ group metabolizes to yield intermediategroup —CR^(d)R^(d)-A-H is not critical, and can be caused by, forexample, hydrolysis under the acidic conditions of the stomach, and/orby enzymes present in the digestive tract and/or tissues or organs ofthe body. Indeed, the R³ group(s) can be selected to metabolize at aparticular site within the body. For example, many esters are cleavedunder the acidic conditions found in the stomach. Prodrugs designed tocleave chemically in the stomach to the active 2,4-pyrimidinediamine canemploy progroups including such esters. Alternatively, the progroups maybe designed to metabolize in the presence of enzymes such as esterases,amidases, lipolases, phosphatases including ATPases and kinase etc., toyield the intermediate group of formula CR^(d)R^(d)-A-H. Progroupsincluding linkages capable of metabolizing in vivo to yield such anintermediate group are well-known, and include, by way of example andnot limitation, ethers, thioethers, silylethers, silylthioethers,esters, thioesters, carbonates, thiocarbonates, carbamates,thiocarbamates, ureas, thioureas, carboxamides, etc. In some instances,a “precursor” group that is oxidized by oxidative enzymes such as, forexample, cytochrome P450 of the liver, to a metabolizable group, can beselected.

The identity of the R³ group can also be selected so as to impart theprodrug with desirable characteristics. For example, lipophilic groupscan be used to decrease water solubility and hydrophilic groups can beused to increase water solubility. In this way, prodrugs specificallytailored for selected modes of administration can be obtained. The R³group can also be designed to impart the prodrug with other properties,such as, for example, improved passive intestinal absorption, improvedtransport-mediated intestinal absorption, protection against fastmetabolism (slow-release prodrugs), tissue-selective delivery, passiveenrichment in target tissues, targeting-specific transporters, etc.Groups capable of imparting prodrugs with these characteristics arewell-known, and are described, for example, in Ettmayer et al., 2004, J.Med. Chem. 47(10:2393-2404), the disclosure of which is incorporated byreference. All of the various groups described in these references canbe utilized in the prodrugs described herein.

In some embodiments, R³ is selected from R^(f), —C(O)R^(f),—C(O)NR^(f)R^(f) and —SiR^(f)R^(f)R^(f), where the R^(f) groups areselected so as to impart the prodrugs with desired bioavailability,cleavage and/or targeting properties. In a specific embodiment, theR^(f) groups are selected to impart the prodrug with higherwater-solubility than the underlying active 2,4-pyrimidinediamine drug.Thus, in some embodiments, the R^(f) groups are selected such that they,taken together with the heteroatom or group to which they are bonded,are hydrophilic in character. Such hydrophilic groups can be charged oruncharged, as is well-known in the art. As specific examples, the R^(f)groups may be selected from hydrogen, optionally substituted loweralkyl, optionally substituted lower heteroalkyl, optionally substitutedlower cycloalkyl, optionally substituted lower heterocycloalkyl,optionally substituted (C6-C10) aryl, optionally substituted 5-10membered heteroaryl, optionally substituted (C7-C18) arylalkyl andoptionally substituted 6-18 membered heteroarylalkyl. The nature of anypresent substituents can vary widely, as is known in the art. In someembodiments any present substituents are, independently of one another,selected from R^(b), defined above.

In a specific embodiment, the progroups on the prodrugs of formula (I)and/or (Ia) are of the formula —CR^(d)R^(d)-A-R³, where R³ is selected

from —(CH₂)_(i)—R^(b), —C(O)R^(a), —C(O)—(CH₂)_(i)—R^(b), —C(O)O—R^(a)and —C(O)O—(CH₂)_(i)—R^(b), where X, R^(a), R^(b) and R^(d) are aspreviously defined, and i is an integer ranging from 0 to 6. Specific,non-limiting, examples of exemplary water-solubility increasingprogroups include by the way of example and not limitation, hydrophilicgroups such as alkyl, aryl, arylalkyl, or cycloheteroalkyl groupssubstituted with one or more of an amine, alcohol, a carboxylic acid, aphosphorous acid, a sulfoxide, a sugar, an amino acid, a thiol, apolyol, a ether, a thioether and a quaternary amine salt.

One important class of progroups includes progroups that contain aphosphate group, for example, phosphate-containing progroups of theformula —(CR^(d)R^(d))_(y)—O—P(O)(OH)₂, where R^(d) is as defined aboveand y is an integer ranging from 1 to 3, typically 1 or 2. In a specificembodiment, each R^(d) is, independently of the others, selected fromhydrogen, substituted or unsubstituted lower alkyl, substituted orunsubstituted (C6-C14) aryl and substituted or unsubstituted (C7-C20)arylalkyl.

While not intending to be bound by any theory of operation, it isbelieved that such phosphate-containing progroups R^(p) act assubstrates for both alkaline and acid phosphatase enzymes, leading totheir removal from the prodrugs under physiological conditions of use.As alkaline phosphatases are abundant in the digestive tract of humans,phosphate-containing progroups R^(p) that can be cleaved in the presenceof alkaline phosphatases are particularly suitable for formulatingphosphate-containing prodrugs intended for oral administration. Specificexamples of phosphate-containing progroups R^(p) suitable for use inprodrugs intended for oral administration include, but are not limitedto, groups of the formula —(R^(d)R^(d))_(y)—O—P(O)(OH)₂ in which eachR^(d) is, independently of the others, selected from hydrogen andunsubstituted lower alkanyl. Exemplary embodiments of suchphosphate-containing progroups include, but are not limited to,—CH₂—O—P(O)(OH)₂ and —CH₂CH₂—O—P(O)(OH)₂.

Although phosphate-containing prodrugs suitable for oral administrationare of interest, skilled artisans will appreciate that prodrugsincluding phosphate-containing progroups R^(p) can be administered viaother routes of administration, as phosphatases are distributedthroughout the body. For example, exemplary prodrug Compound 4 has beenfound to metabolize to the active drug Compound 1 in in vitroexperiments carried out with rat plasma, as well as with rat hepatic andintestinal microsomal preparations, indicating that phosphatases arealso present in plasma. Thus, the only requirement is that theparticular phosphate-containing progroup R^(p) selected should beremovable under the conditions of intended use.

While not intending to be bound by any theory of operation, it isbelieved that when y is 1, phosphate-containing prodrugs, such as thoseaccording to structural formula (Ia), are metabolized to the active2,4-pyrimidinediamine compound via the corresponding hydroxymethylamine.This metabolism is illustrated in FIG. 1A. Referring to FIG. 1A, removalof phosphoric acid from phosphate prodrug 16 via enzymatic hydrolysisyields the corresponding hydroxymethylamine 18, which undergoeshydrolysis in vivo to yield formaldehyde and active2,4-pyrimidinediamine compound 10.

Referring to FIG. 1B, when y is 2, it is believed that in vivohydrolysis of phosphate prodrug 26 yields active 2,4-pyrimidinediamine10 and enol phosphate, which then hydrolyses in vivo to acetaldehyde andphosphoric acid.

Referring again to FIG. 1A, skilled artisan will appreciate that whilehydroxymethylamine 18 metabolizes under physiological conditions toyield active 2,4-pyrimidinediamine compound 10, it is stable at pH 7 andcan therefore be prepared and administered as a hydroxyalkyl-containingprodrug of active compound 10. Thus, in some embodiments of the prodrugsof structural formula (I), R^(p) is a hydroxyalkyl-containing progroupof the formula —CR^(d)R^(d)—OH, where R^(d) is as previously defined. Ina specific exemplary embodiment, R^(p) is —CH₂OH.

Still referring again to FIG. 1A, skilled artisans will also appreciatethat phosphate prodrugs can be generated by in vivo hydrolysis ofphosphate ester prodrugs, such as phosphate ester prodrugs 20 and/or byin vivo oxidation of phosphite prodrugs, such as phosphite prodrugs 24.Such phosphate ester and phosphite prodrugs can in turn be generated byeither in vivo oxidation or hydrolysis of phosphite ester prodrugs suchas phosphite ester prodrugs 22. The corresponding phosphate ester,phosphite and phosphite ester prodrugs of phosphate prodrug 26 areillustrated in FIG. 1B as compounds 30, 34 and 32, respectively. Thus,as will be appreciated by skilled artisans, prodrugs that includeprecursors of phosphates that can metabolize into phosphate groups invivo are also included in the present invention.

In some embodiments of such prodrugs, the phosphorous-containingprogroup R′ comprises a phosphite group. A specific exemplary embodimentof such phosphite-containing prodrugs includes prodrug compounds inwhich the progroup R^(p) is of the formula—(CR^(d)R^(d))_(y)—O—P(OH)(OH), where R^(d) and y are as previouslydefined.

In other embodiments of such prodrugs, the phosphorous-containingprogroup R^(p) comprises an acyclic phosphate ester or phosphite estergroup. Specific exemplary embodiments of such acyclic phosphate esterand phosphite ester prodrugs include progroups R^(p) of the formula—(CR^(d)R^(d))_(y)—O—P(O)(OH)(OR^(e)),—(CR^(d)R^(d))_(y)—O—P(O)(OR^(e))₂, —(CR^(d)R^(d))_(y)—O—P(OH)(OR^(e))and —(CR^(d)R^(d))_(y)—O—P(OR^(e))₂, where R^(e) is selected fromsubstituted or unsubstituted lower alkyl, substituted or unsubstituted(C6-C14) aryl (e.g., phenyl, naphthyl, 4-lower alkoxyphenyl,4-methoxyphenyl), substituted or unsubstituted (C7-C20) arylalkyl (e.g.,benzyl, 1-phenylethan-1-yl, 2-phenylethan-1-yl),—(CR^(d)R^(d))_(y)—OR^(f), —(CR^(d)R^(d))_(y)—O—C(O)R^(f),—(CR^(d)R^(d))_(y)—O—C(O)OR^(f), —(CR^(d)R^(d))_(y)—S—C(O)R^(f),—(CR^(d)R^(d))_(y)—S—C(O)OR^(f), —(CR^(d)R^(d))_(y)—NH—C(O)R^(f),—(CR^(d)R^(d))_(y)—NH—C(O)OR^(f) and —Si(R^(d))₃, wherein each R^(f) is,independently of the others, selected from hydrogen, unsubstituted orsubstituted lower alkyl, substituted or unsubstituted (C6-C14) aryl, andsubstituted or unsubstituted (C7-C20) arylalkyl, and R^(d) and y are aspreviously defined.

In still other embodiments, phosphorous-containing prodrugs that includephosphate precursors are prodrugs in which the phosphorous-containingprogroup R^(p) comprises a cyclic phosphate ester of the formula

where each R^(g) is, independently of the others, selected from hydrogenand lower alkyl; each R^(h) is, independently of the others, selectedfrom hydrogen, substituted or unsubstituted lower alkyl, substituted orunsubstituted lower cycloheteroalkyl, substituted or unsubstituted(C6-C14) aryl, substituted or unsubstituted (C7-C20) arylalkyl andsubstituted or unsubstituted 5-14 membered heteroaryl; z is an integerranging from 0 to 2; and R^(d) and y are as previously defined.

In still other embodiments, phosphorous-containing prodrugs that includephosphate precursors are prodrugs in which the phosphorous-containingprogroup R^(p) comprises a cyclic phosphite ester of the formula

where R^(g), R^(h), R^(d), y and z are as previously defined.

In some embodiments, the substituents R^(h) on such cyclic phosphateester and phosphite ester prodrugs are selected such that the progroupis metabolized in vitro by esterase enzymes. Specific examples of suchphosphate ester and phosphite ester progroups include those in whicheach R^(h) is, independently of the others, selected from hydrogen,lower alkyl, methyl, ethyl and propyl. In some embodiments, suchprogroups are selected from

Many of these phosphate esters and phosphite esters are acid label and,when administered orally, metabolize to the corresponding phosphates andphosphites under the acidic conditions of the stomach and/or gut.

Thus, in the phosphorous-containing prodrugs described herein, theidentity of the particular phosphorous-containing progroups R^(p)employed can be selected to tailor the prodrugs for particular modes ofdelivery, etc.

The suitability of any particular progroup R^(p) for a desired mode ofadministration can be confirmed in biochemical assays. For example, if aprodrug is to be administered by injection into a particular tissue ororgan, and the identities of the various phosphatases expressed in thetissue or organ are known, the particular prodrug can be tested formetabolism in biochemical assays with the isolated phosphatase(s).Alternatively, the particular prodrug can be tested for metabolism tothe active 2,4-pyrimidinediamine compound with tissue and/or organextracts. Using tissue and/or organ extracts can be of particularconvenience when the identity(ies) of the phosphatases expressed in thetarget tissues or organs are unknown, or in instances when the isolatedphosphatases are not conveniently available. Skilled artisans will beable to readily select progroups R^(p) having metabolic properties (suchas kinetics) suitable for particular applications using such in vitrotests. Of course, specific prodrugs could also be tested for suitablemetabolism in in vitro animal models.

In some embodiments, the prodrugs are prodrugs according to structuralformula (I) or (Ia) that have one or more features selected from:

(i) R⁵ is fluoro;

(ii) R² is a phenyl optionally substituted with one or more of the sameor different R⁸ groups;

(iii) R² is 3,4,5-tri(loweralkoxy)phenyl;

(iv) R² is 3,4,5-trimethoxyphenyl;

(v) Y or Y¹ is O; Z¹ is CH, Z² is N; R¹⁷ and R¹⁸ are each methyl; andR¹⁹ and R²⁰ are taken together to form an oxo group; and

(vi) R^(p) is a hydroxyalkyl-containing progroup of the formula —CH₂OH,or a phosphate-containing progroup of the formula—(CR^(d)R^(d))_(y)—O—P(O)(OH)₂, or a phosphate ester, phosphite orphosphite ester analog thereof, wherein y is 1 or 2 and each R^(d) is,independently of the others, selected from hydrogen and unsubstitutedlower alkyl, or

(vii) R′ is selected from —CH₂OH,

CH₂—SH, —CH₂—NH₂, —CH₂—NHR⁵⁰, —CH₂—N(R⁵⁰)₂, —CH₂-A-R^(f),—CH₂-A-C(O)R^(f), —CH₂-A-C(O)OR^(f) and —CH₂-A-C(O)NR^(f)R^(f), where A,R⁵⁰ and R^(f) are as previously defined.

In some embodiments, the prodrugs of structural formulae (I) and (Ia)have two or three of the above-delineated features. In one specificembodiment, the prodrugs have features (i), (iii) and (v). In anotherspecific embodiment, the prodrugs have features (i), (iv) and (v). Instill another specific embodiment, the prodrugs have features (i),(iii), (v) and (vi) or (vii). In still another specific embodiment, theprodrugs have features (i), (iv), (v) and (vi) or (vii). In stillanother specific embodiment, R′ is a phosphate-containing progroup ofthe formula —(CR^(d)R^(d))_(y)—O—P(O)(OH)₂.

In all of the compounds described herein that include substituentalternatives that may be substituted, such as, for example, some of thesubstituent alternatives delineated for R^(d), R^(e), R^(f), R^(g),R^(h), R^(i) and R^(j), the substitutions are typically, independentlyof one another, selected from amongst the R^(b) groups described inconnection with structural formula (I). In a specific embodiment, anypresent substitutions are, independently of one another, selected fromhydroxyl, lower alkoxy, (C6-C14) aryloxy, lower alkoxyalkyl,methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl and halogen.

Those of skill in the art will appreciate that many of the prodrugsdescribed herein, as well as the various prodrug species specificallydescribed and/or illustrated herein, may exhibit the phenomena oftautomerism, conformational isomerism, geometric isomerism and/oroptical isomerism. For example, the prodrugs may include one or morechiral centers and/or double bonds and as a consequence may exist asstereoisomers, such as double-bond isomers (i.e., geometric isomers),enantiomers and diasteromers and mixtures thereof, such as racemicmixtures. As another example, the prodrugs may exist in severaltautomeric forms, including the enol form, the keto form and mixturesthereof. As the various compound names, formulae and drawings within thespecification and claims can represent only one of the possibletautomeric, conformational isomeric, optical isomeric or geometricisomeric forms, it should be understood that the invention encompassesany tautomeric, conformational isomeric, optical isomeric and/orgeometric isomeric forms of the prodrugs having one or more of theutilities described herein, as well as mixtures of these variousdifferent isomeric forms. In cases of limited rotation around the2,4-pryimidinediamine moiety, atrop isomers are also possible and arealso specifically included in the compounds of the invention.

Moreover, skilled artisans will appreciate that when lists ofalternative substituents include members which, owing to valencyrequirements or other reasons, cannot be used to substitute a particulargroup, the list is intended to be read in context to include thosemembers of the list that are suitable for substituting the particulargroup. For example, skilled artisans will appreciate that while all ofthe listed alternatives for R^(b) can be used to substitute an alkylgroup, certain of the alternatives, such as ═O, cannot be used tosubstitute a phenyl group. It is to be understood that only possiblecombinations of substituent-group pairs are intended.

The prodrugs described herein may be identified by either their chemicalstructure or their chemical name. When the chemical structure and thechemical name conflict, the chemical structure is determinative of theidentity of the specific prodrug.

Depending upon the nature of the various substituents, the prodrugsdescribed herein may be in the form of salts. Such salts include saltssuitable for pharmaceutical uses (“pharmaceutically-acceptable salts”),salts suitable for veterinary uses, etc. Such salts may be derived fromacids or bases, as is well-known in the art.

In one embodiment, the salt is a pharmaceutically acceptable salt.Generally, pharmaceutically acceptable salts are those salts that retainsubstantially one or more of the desired pharmacological activities ofthe parent compound and which are suitable for administration to humans.Pharmaceutically acceptable salts include acid addition salts formedwith inorganic acids or organic acids. Inorganic acids suitable forforming pharmaceutically acceptable acid addition salts include, by wayof example and not limitation, hydrohalide acids (e.g., hydrochloricacid, hydrobromic acid, hydriodic, etc.), sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids suitable for formingpharmaceutically acceptable acid addition salts include, by way ofexample and not limitation, acetic acid, trifluoroacetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalicacid, pyruvic acid, lactic acid, malonic acid, succinic acid, malicacid, maleic acid, fumaric acid, tartaric acid, citric acid, palmiticacid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid,mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid,ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonicacid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, etc.),4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like.

Pharmaceutically acceptable salts also include salts formed when anacidic proton present in the parent compound is either replaced by ametal ion (e.g., an alkali metal ion, an alkaline earth metal ion or analuminum ion) or coordinates with an organic base (e.g., ethanolamine,diethanolamine, triethanolamine, N-methylglucamine, morpholine,piperidine, dimethylamine, diethylamine, etc.).

The prodrugs described herein, as well as the salts thereof, may also bein the form of hydrates, solvates and N-oxides, as are well-known in theart. Unless specifically indicated otherwise, the expression “prodrug”is intended to encompass such salts, hydrates, solvates and/or N-oxides.Specific exemplary salts include, but are not limited to, mono- anddi-sodium salts, mono- and di-potassium salts, mono- and di-lithiumsalts, mono- and di-alkylamino salts, mono-magnesium salts, mono-calciumsalts and ammonium salts.

Another aspect of the invention provides a prodrug salt, i.e., a salt ofa prodrug compound described above. For example, one embodiment of theinvention provides a produg salt, or a hydrate, solvate or N-oxidethereof, comprising a 2,4-pyrimidineamine moiety and at least oneprogroup salt R^(q) linked covalently to a primary or secondary aminonitrogen atom of the 2,4-pyrimidinediamine moiety.

The progroup salt R^(q) may be any suitable salt of any acidic progroupR^(p). For example, the progroup salt R^(q) may be a salt of aphosphate-containing progroup, a carbonate-containing progroup, or asulfonate-containing progroup. The counterion may be, for example, analkali cation (e.g., Na⁺), an alkaline earth cation (e.g. [Ca²⁺]_(0.5)),or an ammonium cation (e.g., NH₄ ⁺). Examples of suitable progroup saltsR^(q)

include: —(CR^(d)R^(d))_(y)-A-P(O)(O⁻)₂M⁺ ₂,—(CR^(d)R^(d))_(y)-A-P(O)(OR^(e))(O⁻)M⁺, —(CR^(d)R^(d))_(y)-A-P(O)(OH)(O⁻) M⁺, —(CR^(d)R^(d))_(y)-A-P(O)₂M⁺ ₂,—(CR^(d)R^(d))_(y)-A-P(OR^(e))(O⁻)M⁺, —(CR^(d)R^(d))_(y)-A-P(OH)(O⁻)M⁺,and —(CR^(d)R^(d))_(y)-A-COO⁻M⁺, wherein A, R^(d), R^(e), and y are asdescribed above and M⁺ is as described below. In one embodiment of theinvention, A is O.

In one aspect of the invention, the prodrug salt has the structuralformula (IX),

in which Y, Z¹, Z², R², R⁵, R⁸, R¹⁷, R¹⁸, R¹⁹, R²⁰, R^(a), R^(b), R^(c)and R²⁴ are as described above for formula (I),

R²¹, R²² and R²³ are each, independently of one another, selected fromhydrogen, a progroup R^(p) and a progroup salt R^(q);

R²⁴ is selected from hydrogen, lower alkyl, a progroup R^(p) and aprogroup salt R^(q);

each m is, independently of the others, an integer from 1 to 3; and

each n is, independently of the others, an integer from 0 to 3, with theproviso that at least one of R²¹, R²², R²³ and R²⁴ is a progroup saltR^(q).

As described above in connection with formula (Ia), in certainembodiments of the invention the prodrug salt includes only one progroupsalt R^(q). A specific embodiment of prodrug salts that include a singleprogroup salt R^(q) are compounds according to structural formula (IXa):

wherein Y¹ is selected from CH₂, NR²⁴, O, S, S(O) and S(O)₂; and Z², R²,R⁵, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²⁴ and R^(q) are as previously defined, withthe proviso that R² does not include any R^(p) or R^(q) groups.

In some embodiments, the prodrug salts are prodrug salts according tostructural formula (IX) or (IXa) that have one or more features selectedfrom:

(i) R⁵ is fluoro;

(ii) R² is a phenyl optionally substituted with one or more of the sameor different R⁸ groups;

(iii) R² is 3,4,5-tri(loweralkoxy)phenyl;

(iv) R² is 3,4,5-trimethoxyphenyl;

(v) Y or Y¹ is O; Z¹ is CH, Z² is N; R¹⁷ and R¹⁸ are each methyl; andR¹⁹ and R²⁰ are taken together to form an oxo group; and

(vi) R^(q) is a phosphate-containing progroup salt of the formula—(CR^(d)R^(d))_(y)—O—P(O)(O⁻)₂M⁺ ₂, or a phosphate monoester, phosphiteor phosphite monoester analog thereof, wherein y is 1 or 2 and eachR^(d) is, independently of the others, selected from hydrogen andunsubstituted lower alkyl, or

(vii) R^(q) is —CH₂-A-C(O)O⁻M⁺.

In some embodiments of the invention, the prodrug salts of structuralformulae (IX) and (IXa) have two or three of the above-delineatedfeatures (i)-(vii). In one specific embodiment, the prodrugs havefeatures (i), (iii) and (v). In another specific embodiment, theprodrugs have features (i), (iv) and (v). In still another specificembodiment, the prodrugs have features (i), (iii), (v) and (vi) or(vii). In still another specific embodiment, the prodrugs have features(i), (iv), (v) and (vi) or (vii). In still another specific embodiment,R^(q) is a phosphate-containing progroup salt of the formula—(CR^(d)R^(d))_(y)—O—P(O)(O⁻)₂M⁺ ₂. In another embodiment of theinvention, a prodrug salt of formula (IXa) has features (iv) and (v) andthe progroup salt R^(q) is a salt of —(CR^(d)R^(d))_(y)—O—P(O)(O⁻)₂,where y is an integer ranging from 1 to 3; each R^(d) is, independentlyof the others, selected from hydrogen, optionally substituted loweralkyl, optionally substituted (C6-C14) aryl and optionally substituted(C7-C20) arylalkyl; where the optional substituents are, independentlyof one another, selected from hydroxyl, lower alkoxy, (C6-C14) aryloxy,lower alkoxyalkyl, methoxymethyl, methoxyethyl, ethoxymethyl,ethoxyethyl and halogen, or, alternatively, two R^(d) bonded to the samecarbon atom are taken together with the carbon atom to which they arebonded to form a cycloalkyl group containing from 3 to 8 carbon atoms.Alternatively, a prodrug salt of formula (IXa) has features (iv) and(v), and the progroup salt R^(q) is selected from —CH₂—O—P(O)(O⁻)₂M⁺ ₂and —CH₂CH₂—O—P(O)(O⁻)₂M⁺ ₂.

Another aspect of the invention provides prodrug salts having thestructure (X):

wherein Y₁, Z₂, R₅, R₁₇, R₁₈, R₁₉ and R₂₀ are as described above, R³⁰,R³¹ and R³² are each, independently of one another, selected fromhydrogen, lower alkyl, lower alkenyl, lower alkynyl, (C6-C14) aryl,phenyl, 5-14 membered heteroaryl, (C7-C20) arylalkyl, benzyl, 7-20membered heteroarylalkyl, —OR, chloro, fluoro, bromo, cyano,nitro, —C(O)R, —C(O)OR, —NRR, —S(O)₂NRR, —C(O)NRR, —N(R)S(O)₂R and—NC(O)OR, where each R is, independently of the others, selected fromhydrogen and lower alkyl; andR^(q) is selected from —CH₂—O—P(O)(O⁻)₂M⁺ ₂, —CH₂CH₂—O—P(O)(O⁻)₂M⁺ ₂, —CH₂—O—P(O)(OH)(O⁻)M⁺, and —CH₂CH₂—O—P(O)(OH)(O⁻)M⁺. In oneembodiment of the invention, R³⁰, R³¹ and R³² are each methoxy.

Another aspect of the invention relates to prodrug salts having thestructure (XI):

in which R^(q) is —CH₂—O—P(O)(O⁻)₂M⁺ ₂, —CH₂CH₂—O—P(O)(O⁻)₂M⁺ ₂,—CH₂—O—P(O)(OH)(O⁻)M⁺, or —CH₂CH₂—O—P(O)(OH)(O⁻)M⁺, and hydratesthereof. M⁺ may be, for example, an alkali cation, an alkaline earthcation, or an ammonium cation (e.g., a lysine cation or an argininecation). For example, according to certain embodiments of the invention,R^(q) may be is —CH₂—O—P(O)(O⁻)₂Na⁺ ₂; —CH₂—O—P(O)(OH)(O⁻)Na⁺;—CH₂—O—P(O)(O⁻)₂K⁺ ₂; —CH₂—O—P(O)(OH)(O⁻) K⁺; —CH₂—O—P(O)(O⁻)₂Ca²⁺;—CH₂—O—P(O)(OH)(O⁻)[Ca²⁺]_(0.5); —CH₂—O—P(O)(O⁻)₂Mg²⁺; or—CH₂—O—P(O)(OH)(O⁻)[Mg²⁺]_(0.5). In certain embodiments of theinvention, M+ is an alkali cation, an amino acid cation, or an ammoniumcation. In certain embodiments of the invention, the prodrug salts arein solid or semi-solid form, and are not dissolved in aqueous solution.

Another aspect of the invention relates to hydrates of the prodrug saltsdescribed above. Hydrates desirably have, for example, ranging fromabout 1 to about 15 moles of water per mole of prodrug salt. As isdescribed in more detail below, the inventors have determined thatprodrug salt hydrates of the present invention have surprisinglydesirable stability characteristics, remaining at a stable level ofhydration over wide ranges of relative humidities. The inventors surmisethat the hydration stability characteristics are due to the high bindingenergy of the water molecules to the hydrate as well as the specialstability of the three dimensional order of the crystalline matrix andits inability to spatially rearrange itself in response to dehydration.

According to one aspect of the invention, prodrug salt hydrates of theinvention contain from about 3% to about 17% by weight of water, morepreferably from about 13.0% to 16.5% by weight of water. In oneembodiment, prodrug salt hydrates of the present invention have theformula

in which x is from about 1 to about 15. More preferably x is from about5 to about 10. In one particular embodiment, x is from about 5 to about8. For example, in one embodiment the prodrug salt hydrate of thepresent invention has the structure:

Control of pH in the formation of the salts is desirable. Desirable pHvalues for the formation of di-M⁺ salts of compound 4 range from about 8to about 11, more preferably from about 9 to about 11, even morepreferably from about 9.3 to about 10.5. For example, desirable pHvalues for the formation of the disodium salt of compound 4 fall withinthese ranges. Desirable pH values for the formation of mono-M⁺ (e.g.,monosodium) salts of compound 4 range from about 5 to about 7, morepreferably from about 5 to about 6, even more preferably from about 5.0to about 5.5.

One embodiment of the invention provides a method for the preparation ofa prodrug salt hydrate having the formula

the method comprising precipitating the prodrug salt hydrate from anaqueous solution of the prodrug

and an [M⁺]-containing base, the aqueous solution having a pH value inthe range of about 9 to about 11. In some embodiments the range is fromabout 9.3 to about 10.5.

In another embodiment, the invention provides a method for thepreparation of a prodrug salt hydrate

the method comprising precipitating the prodrug salt hydrate from anaqueous solution of the prodrug

and a sodium-containing base, the aqueous solution having a pH a valuein the range of about 9 to about 11. In some embodiments the pH range isfrom about 9.3 to about 10.5.

One embodiment of the invention provides a method for the preparation ofa prodrug salt hydrate having the formula

the method comprising adjusting the pH of an aqueous solution of theprodrug

to a value in the range of about 9 to about 11 with an [M⁺]-containingbase. In some embodiments the pH is adjusted to a value from about 9.3to about 10.5.

In another embodiment, the invention provides a method for thepreparation of a prodrug salt hydrate

the method comprising adjusting the pH of an aqueous solution of theprodrug

to a value in the range of about 9 to about 11 with a sodium-containingbase. In some embodiments the pH is adjusted to a value from about 9.3to about 10.5.

One embodiment of the invention provides a method for the preparation ofa prodrug salt hydrate having the formula

the method comprising:

-   -   (i) adjusting the pH of an aqueous solution of the prodrug

to a value in the range of about 9 to about 11 with an [M⁺]-containingbase; and

-   -   (ii) precipitating the prodrug salt hydrate from the aqueous        solution. In some embodiments the pH is adjusted to a value from        about 9.3 to about 10.5.

In another embodiment, the invention provides a method for thepreparation of a prodrug salt hydrate

the method comprising:

-   -   (i) adjusting the pH of an aqueous solution of the prodrug

-   -   to a value in the range of about 9 to about 11 with a        sodium-containing base; and    -   (ii) precipitating the prodrug salt hydrate from the aqueous        solution. In some embodiments the pH is adjusted to a value from        about 9.3 to about 10.5.

In some embodiments of the invention precipitating a prodrug salt froman aqueous solution comprises adding an appropriate water-misciblesolvent to the aqueous solution. Alternatively, precipitation can beaccomplished via salt formation in, or salt addition to, awater/water-miscible solvent mixture to dissolve a prodrug salt,followed by precipitation to provide the prodrug salt hydrate. Theaddition of a water-miscible solvent to an aqueous prodrug salt solutioncan hasten the precipitation of a prodrug salt. A water-miscible solventcan be partially water miscible or wholly water miscible. Awater-miscible solvent may be a mixture of one or more solvents, whereinone or more of the solvents is water-miscible.

Prodrug salts of the invention may be precipitated at elevatedtemperatures, ambient temperature, or at sub-ambient temperatures. Inone embodiment, the salt is precipitated at ambient temperature. Inanother embodiment, the salt is precipitated from a warmed solution,either directly or upon cooling. In a specific embodiment, heat isapplied to the salt solution prior to precipitation. Heat can be appliedto the solution directly or by adding a warmed water-miscible solvent tothe aqueous solution. In one embodiment, the prodrug salt solution isheated just before and/or just after adding the water miscible solvent,then allowed to cool to precipitate the salt of the prodrug.

Another embodiment of the invention provides a method for thepreparation of a prodrug salt hydrate

the method comprising:

-   -   (i) adjusting the pH of an aqueous solution of the prodrug

-   -    to a value in the range of about 9 to about 11 with a        sodium-containing base;    -   (ii) adding a water-miscible solvent to the aqueous solution;    -   (iii) heating the aqueous solution before adding the solvent,        after adding the water-miscible solvent, or both;    -   (iv) cooling the aqueous solution to precipitate the prodrug        salt hydrate. In some embodiments the pH is adjusted to a value        from about 9.3 to about 10.5.

In some embodiments of the invention, one of the above-described methodsfurther comprises isolating the prodrug salt hydrate from the solution.Isolating the prodrug salt hydrate may include, for example, washing theprodrug salt hydrate with at least one wash solvent. Thereafter,substantially all of the wash solvent is removed from prodrug salthydrate.

In another embodiment, the invention provides a method for thepreparation of a prodrug salt hydrate

the method comprising:

-   -   (i) adjusting the pH of an aqueous solution of the prodrug

-   -    to a value in the range of about 9 to about 11 with a        sodium-containing base;    -   (ii) adding a water-miscible solvent to the aqueous solution;    -   (iii) heating the aqueous solution, before adding the water        miscible solvent, after adding the water-miscible solvent, or        before and after adding the water-miscible solvent;    -   (iv) cooling the aqueous solution to allow precipitation of the        prodrug salt hydrate; and    -   (v) washing the prodrug salt hydrate with at least one wash        solvent. In some embodiments the pH is adjusted to a value from        about 9.3 to about 10.5.

In some embodiments of the invention, the prodrug salt isolated (e.g.,from salt formation in situ and precipitation) is not a desired hydrateform and/or crystalline form. For example, a solid prodrug salt of theinvention can be amorphous (solvated or not) or crystalline (solvated ornot).

Another aspect of the invention relates to methods of preparation ofcrystalline forms of hydrates of the prodrug salts described above. Inthese embodiments, the prodrug salt form is further manipulated toconvert it to the desired prodrug salt hydrate and/or crystalline form.For example, in one embodiment, a prodrug salt is isolated andthereafter exposed to conditions under which the solid converts to adesired crystalline and/or hydrate form. For example, using methodsdescribed herein, a prodrug salt can be isolated in a crystalline formhaving less than a desirable amount of water. This crystalline form isexposed to, for example, a moisture-containing environment (e.g., humidor moist atmosphere) to convert it to a desirable hydrate form. Inanother example, a prodrug salt can be isolated in amorphous form, forexample via removing solvent from a solution of the salt under vacuum.The amorphous salt is exposed to a moist atmosphere and is therebyconverted over time to a crystalline hydrate.

Depending upon the composition, handling, and isolation techniques,prodrug salts of the invention may be crystalline or amorphous. Also,the aforementioned factors may dictate the water content of the saltform. As mentioned, prodrug salt hydrates of the invention preferablycontain from about 3% to about 17% by weight of water, more preferablyfrom about 13.0% to 16.5% by weight of water. In a preferred embodiment,a prodrug having the formula,

in which x is from about 1 to about 15, is precipitated from an aqueoussolution using methods described herein. The prodrug hydrate is isolatedas a crystalline or amorphous form. The prodrug hydrate is exposed to amoist environment which over time converts the prodrug hydrate to acrystalline form of desired hydration level. Moist environments include,for example, moist air or moist inert gas; generally “moisture” is usedto describe environments having a sufficient degree of moisture toresult in hydration of the anhydrous prodrug salt or increased hydrationof the prodrug salt hydrate. In a particularly preferred embodiment, thecrystalline prodrug hydrate is the disodium hexahydrate form.

In another embodiment, the invention provides a method for thepreparation of a crystalline form of the prodrug salt hydrate

the method comprising:

-   -   (i) adjusting the pH of an aqueous solution of the prodrug

-   -    to a value in the range of about 9 to about 11 with a        sodium-containing base;    -   (ii) adding a water-miscible solvent to the aqueous solution;    -   (iii) heating the aqueous solution, before adding the water        miscible solvent, after adding the water-miscible solvent, or        before and after adding the water-miscible solvent;    -   (iv) precipitating a prodrug salt from the aqueous solution via        cooling;    -   (v) washing the prodrug salt with at least one wash solvent;    -   (vi) removing substantially all of the wash solvent from the        prodrug salt; and    -   (vii) exposing the prodrug salt to moisture, yielding the        crystalline form of the prodrug salt hydrate.

In some embodiments the pH is adjusted to a value from about 9.3 toabout 10.5. Moist air is typically used as a source of moisture.

In the methods described above, the water-miscible solvent may be, forexample, acetonitrile, acetone, isopropanol, methanol or ethanol. Incertain embodiments of the methods described above, the water-misciblesolvent is isopropanol. As mentioned, the water-miscible solvent may bea mix of two or more solvents, wherein at least one is itselfwater-miscible.

In the methods described above, the wash solvent may be, for example,isopropanol, methanol, ethanol, acetone, or acetonitrile, or mixturesthereof. In certain embodiments of the methods described above, theprecipitated crystals are washed with two wash solvents.

In one embodiment, the invention provides a method for the preparationof a crystalline form of the prodrug salt hydrate

the method comprising

adjusting the pH of an aqueous solution of the prodrug

to a value in the range of about 9 to about 11 with an [M⁺]-containingbase;

precipitating a prodrug salt from the aqueous solution; and

exposing the prodrug salt to moisture.

In some embodiments the pH is adjusted to a value from about 9.3 toabout 10.5. Moist air is typically used as a source of moisture.

Methods according to this embodiment of the invention may be performedsubstantially as described above. For example, according to oneembodiment of the invention, a method for making a crystalline form ofthe prodrug salt hydrate

comprises:

(i) adjusting the pH of an aqueous solution of the prodrug

-   -   to a value in the range of about 9 to about 11 with a        sodium-containing base;

(ii) precipitating a prodrug salt from the aqueous solution; and

(iii) exposing the prodrug salt to moisture.

In some embodiments the pH is adjusted to a value from about 9.3 toabout 10.5. Moist air is typically used as a source of moisture.

In a preferred embodiment, the invention provides the method for thepreparation of a crystalline form of the prodrug salt hydrate

the method comprising the:

-   -   (i) adjusting the pH of an aqueous solution of the prodrug

-   -    to a value in the range of about 9 to about 11 with NaOH;    -   (ii) adding isopropanol to the aqueous solution    -   (iii) heating the aqueous solution, before adding isopropanol,        after adding isopropanol, or before and after adding        isopropanol;    -   (iv) cooling the aqueous solution to allow precipitation of a        prodrug salt;    -   (iv) washing the prodrug salt with isopropanol and acetone;    -   (v) removing substantially all of the solvent from the prodrug        salt; and    -   (vi) exposing the prodrug salt to moisture.

In some embodiments the pH is adjusted to a value from about 9.3 toabout 10.5. Moist air or moist inert gas is typically used as a sourceof moisture.

In the methods described above, the heating steps may be performed, forexample, at temperatures above about 50° C.; above about 75° C.; orabove about 90° C. In certain embodiments of the invention, the heatingstep is performed at about the boiling point of the solution.

In the methods described above, the moist air or moist inert gaspreferably has a relative humidity of at least about 10%. In embodimentsof the methods described above the moist air or moist inert gas has arelative humidity of at least about 20%. In other embodiments of themethods described above, the moist air or moist inert gas has a relativehumidity of at least about 30%. In certain embodiments of the methodsdescribed above, the moist air or moist inert gas has a relativehumidity from about 30% to about 60%.

In the methods described above, the [M⁺]-containing base may be, forexample, a hydroxide, an alkoxide or a carbonate. In the methodsdescribed above, the sodium-containing base may be, for example, NaOH,NaOMe, or Na₂CO₃. In certain embodiments, the sodium-containing base isNaOH.

Another aspect of the invention relates to methods for making forms ofmono-M⁺ (e.g., monosodium) salts of the present invention. In oneembodiment of the invention, the invention provides a method for thepreparation of a prodrug salt hydrate of the formula

the method comprising:

-   -   (i) adjusting the pH of an aqueous solution of the prodrug

-   -    to a value in the range of about 5 to about 6 with an        [M⁺]-containing base; and    -   (ii) precipitating the prodrug salt hydrate from the aqueous        solution.

In some embodiments the pH is adjusted to a value from about 5.0 toabout 5.5.

The method for making the mono-M⁺ salt may be modified as describedabove for the di-M⁺ salt and the disodium salts. For example, in oneembodiment, the invention provides a method for the preparation of aprodrug salt hydrate

comprising:

-   -   (i) adjusting the pH of an aqueous solution of the prodrug

-   -    to a value in the range of about 5 to about 6 with a        sodium-containing base;    -   (ii) adding a water-miscible solvent to the aqueous solution;    -   (iii) heating the aqueous solution, before adding the water        miscible solvent, after adding the water-miscible solvent, or        before and after adding the water-miscible solvent; and    -   (iv) cooling the aqueous solution to precipitate the prodrug        salt hydrate.

In some embodiments, the pH is adjusted to a value in the range of about5.0 to about 5.5 with a sodium-containing base.

Another aspect of the invention provides methods of making crystallinehydrate forms of prodrug mono-M⁺ salts. In one embodiment, an amorphousor crystalline form of the mono-M⁺ salt is isolated using in situformation and precipitation as described above. The mono-M⁺ salt isthereafter exposed to conditions that convert it to a preferred hydratedcrystalline form. In a specific example, the mono-M⁺ salt is exposed tomoist air to convert it to a preferred hydrated crystalline form.

Another embodiment of the invention provides a method of preparing acrystalline form of a prodrug salt hydrate having the formula

the method comprising(i) adjusting the pH of a n aqueous solution of the prodrug

to a value in the range of about 5 to about 7 with an [M⁺]-containingbase;(ii) precipitating a prodrug salt from the solution; and(iii) exposing the prodrug salt to moisture, yielding the crystallineform of the prodrug salt hydrate.

Another aspect of the invention provides a pharmaceutical compositioncomprising a prodrug salt hydrate as prepared by any of the methodsdescribed above and a pharmaceutically acceptable carrier, diluentand/or excipient.

Methods of Synthesis

The prodrugs and prodrug salts described herein, as well asintermediates therefor, may be synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods. Suitableexemplary methods that may be routinely used and/or adapted tosynthesize active 2,4-pyrimidinediamine compounds can be found in U.S.Pat. No. 5,958,935, U.S. application Ser. No. 10/355,543 filed Jan. 31,2003 (US2004/0029902A1), international application Serial No.PCT/US03/03022 filed Jan. 31, 2003 (WO 03/063794), U.S. application Ser.No. 10/631,029 filed Jul. 29, 2003 (US 2005/0028212), internationalapplication Serial No. PCT/US03/24087 (WO2004/014382), U.S. applicationSer. No. 10/903,263 filed Jul. 30, 2004 (US2005/0234049), andinternational application Serial No. PCT/US2004/24716 (WO 2005/016893),the disclosures of which are incorporated herein by reference. Theseactive 2,4-pyrimidinediamine compounds can be used as starting materialsto synthesize the prodrugs. Specific examples describing the synthesisof phosphate prodrug Compound 4, as well as a synthetic intermediatetherefor, are provided in the Examples section. All of the prodrugsdescribed herein may be synthesized by routine adaptation of thismethod.

For example, some embodiments of prodrugs according to structuralformula (I) and/or (Ia) can be prepared by reacting the correspondingactive 2,4-pyrimidinediamine (i.e., compounds according to structuralformulae (I) and/or (Ia) in which each R^(p) is hydrogen) with analdehyde or a ketone to give an α-hydroxymethyl amine, which can then bereacted with an electrophile to yield a prodrug. An exemplary synthesisof this type is illustrated in Scheme (I), below:

In Scheme (I), Y¹, Z¹, Z², R², R⁵, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are as definedfor structural formula (I) or (Ia). R³ and R^(d) are as defined in thetext, supra. According to Scheme (I), active 2,4-pyrimidinediamine 10 isreacted with ketone 12 to yield a mixture of four products: unreactedstarting material 10 (not illustrated) and compounds 14a, 14b and 14c.At this stage, the products can be isolated from one another usingstandard chromatographic techniques. Reaction with electropholic R³yields prodrugs 15a, 15b and 15c.

As illustrated above, α-hydroxymethylamines 14a, 14b and 14c can beconverted into a variety of different types of prodrugs 15a, 15b and15c. For example, the α-hydroxymethylamines can be reacted with analcohol in the presence of a strong acid catalyst, or a carbon-bearinghalide (e.g., CH₃Br), to yield the corresponding ether derivatives(e.g., compounds in which R³ is R^(f), where R^(f) is as previouslydefined).

Reacting α-hydroxymethylamines 14a, 14b and 14c with a carboxylic acidin the presence of a strong acid catalyst or a carboxylic acid anhydrideor a carboxylic acid halide (e.g. with an appropriate acid scavenger)yields the corresponding ester derivatives (e.g., compounds in which R³is —C(O)R^(f), where R^(f) is as defined above).

Reaction of α-hydroxymethylamines 14a, 14b and 14c with a haloformateester (e.g., Cl—C(O)OCH₃) yields the corresponding carbonate derivatives(e.g., compounds in which R³ is —C(O)OR^(f), where R^(f) is aspreviously defined).

Reaction of α-hydroxymethylamines 14a, 14b and 14c with a haloformamide(e.g., Cl—C(O)N(CH₃)₂) yields the corresponding carbamate or urethanederivatives (e.g., compounds in which R³ is —C(O)NR^(f)R^(f), whereR^(f) is as previously defined).

As will be recognized by skilled artisans, other hydroxyl protectinggroups could also be used, including, for example, the various differenthydroxyl protecting groups described in Green & Wuts, “Protective Groupsin Organic Chemistry,” 2d Edition, John Wiley & Sons, New York, pp.10-142, the disclosure of which is incorporated herein by reference.

Alternatively, prodrugs according to structural formulae (I) and (Ia)can be synthesized by nucleophilic substitution of the correspondingphosphate esters. An example of this synthetic route is illustrated inScheme (II), below:

According to Scheme (II), active 2,4-pyrimidinediamine 10 is reactedwith di-tert-butyl chloromethylphosphate 13 in the presence of cesiumcarbonate to yield a mixture of four products: unreacted startingmaterial 10 (not illustrated) and phosphate esters 17a, 17b and 17c,which are themselves prodrugs as described herein. When R² is3,4,5-trimethoxyphenyl phosphate ester 17a is the major product.Reaction of this phosphate ester 17a with R³-AH (where A is O, S, orNR⁵⁰), yields prodrug 19. The minor phosphate esters 17b and 17c can besimilarly reacted to yield the corresponding prodrugs.

Di-tert-butyl chloromethyl phosphate 13 can be prepared fromdi-tert-butyl phosphate as illustrated in Scheme (III), below:

According to Scheme (III), di-tert-butyl phosphate 9 is obtained fromthe corresponding di-tert-butyl phosphite 7 as described in Krise etal., 1990, J. Med. Chem. 42:3793-3794. Reaction of phosphate 9 withchloromethyl chlorosulfate 11 (available from Synergetica, Inc.,Sicklerville, N.J. 08081) as described in Mantyla et al., 2002, Tet.Lett. 43:3793-3794 yields di-tert-butyl chloromethyl phosphate 13, whichcan be used in Scheme (II), above, crude without purification.

Although the Schemes illustrated above depict the synthesis of prodrugsthat include a single progroup, prodrugs having a plurality of progroupscould be obtained by adjusting the number of equivalents of reagent 12or 13 used.

As another alternative to Scheme (I), hydroxymethylamine 14a can beprepared in a two-step process by first reacting active2,4-pyrimidinediamine 10 with a bis functional electrophile, such as,for example, chloro-iodomethane (I—CH₂Cl), to yield a chloro-methylintermediate, which can then be hydroxylated by reaction with basichydroxide or reacted with various nucleophilic reagents such asalkoxides, amines or sulfide to make R^(p). Specific conditions forcarrying out reactions of this type that can be used to synthesize theprodrugs described herein, for example, in Bansal et al., 1981, J.Pharm. Sci. 70(8):850-854 and Bansal et al., 1981, J. Pharm. Sci.70(8):855-857, the disclosures of which are incorporated herein byreference.

An exemplary synthetic route that can be used to synthesize an exemplaryphosphate prodrug 16 according to structural formula (Ia) is illustratedin Scheme (IV), below. This method may be routinely adapted tosynthesize the full range of phosphate prodrugs described herein.

In Scheme (IV), Y¹, Z¹, Z², R², R⁵, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are as definedfor structural formula (I) or (Ia). According to Scheme (IV), active2,4-pyrimidinediamine 10 is reacted with di-tert-butylchloromethylphosphate 13 in the presence of cesium carbonate to yield amixture of four products: unreacted starting material 10 (notillustrated) and compounds 17a, 17b and 17c. When R² is3,4,5-trimethyoxyphenyl, compound 17a is the major product. At thisstage, the major product can be isolated from the minor products usingstandard chromatographic techniques. Removal of the tert-butyl groupsyields a mixture of desired product 16 and impurities 18 and 10. Thedesired product 16 can be isolated using standard techniques.

An alternative method of obtaining phosphate prodrug 16 is illustratedin Scheme (V); below.

According to Scheme (V), the reaction of active 2,4-pyrimidinediamine 10again yields a mixture of four products: unreacted pyrimidinediamine 10(not illustrated) major product 17a and minor products 17b and 17c.Major product 17a can be isolated via crystallization (see the Examplessection for suitable conditions), dissolved in a mixture of acetic acidand water (4:1 AcOH:H₂O) and heated to 65° C. for approximately 3 hr toyield phosphate prodrug 16 as the major product.

Although Schemes (IV) and (V) illustrate the synthesis of a phosphateprodrug in which the phosphate progroup is —CH₂—O—P(O)(OH)₂, skilledartisans will appreciate that phosphate prodrugs including otherphosphate progroups could be readily obtained according to the samemethods by using the appropriate reagent 13. Phosphate ester prodrugs,phosphite prodrugs and phosphite ester prodrugs can also be synthesizedvia routine adaptation of the methods using the appropriate phosphateester, phosphite and phosphite ester halides 13. Exemplary methods forsynthesizing cyclic phosphate ester prodrugs, which can be used asprodrugs in the various methods described herein, or converted intophosphate prodrugs, are illustrated in FIG. 3. Moreover, while Schemes(I) and (III) depict compound 16 as being the desired product, prodrugshaving progroups at other positions within the prodrug molecule could bereadily obtained by isolating, for example minor product 17a or 17band/or by adjusting the number of equivalents of reagent 13 used.

Referring to FIG. 3, diols 21 are converted to the corresponding cyclicphosphates 23 using literature procedures as depicted. Cyclic phosphates23 are converted to the corresponding chloromethyl phosphate esters 25in any of the three ways depicted. Compound 1 is converted to cyclicphosphate ester derivatives 27, 29, and 31, via addition of 25 underconditions as previously described for the synthesis of compounds 17a-c.Cyclic phosphate ester derivatives 27, 29, and 31, are converted to thecorresponding phosphate derivatives via treatment under acidicconditions as described for the synthesis of compound 16, or viahydrogenation using, for example, palladium catalyst.

Prodrug salts may be prepared in a variety of ways familiar to theskilled artisan, or according to the methods described herein. Forexample, a prodrug bearing an acidic prodrug group R^(p) may beconverted to the corresponding prodrug salt by treatment with a base,followed by isolation through, for example, concentration,precipitation, or crystallization. If there exists more than one acidichydrogen in the prodrug, then polyanionic salts may be prepared with theappropriate stoichiometric ratio of base and prodrug. For example, adianion may be formed from a prodrug containing a —P(O)(OH)₂ group withtwo equivalents of an appropriate base. Preferred salts of prodrugs ofthe invention include, but are not limited to, sodium, potassium,calcium, magnesium, ammonium, and the like. Alternatively, an organicbase may be used to prepare salts of the prodrugs of the invention.Examples of preferred organic bases include, but are not limited to,lysine, arginine, N,N-diethylethanolamine, diethanolamine, ethanolamine,ethylenediamine, meglumine, morpholine, piperazine, piperidine,triethylamine, tromethamine (Tris), benzathine, benzene-ethanamine,adenine, cytosine and guanidine. As described in more detail below, theprodrug salts may be formed in aqueous solution. Alternatively, they maybe made in organic solution using an organic base such as sodiummethoxide.

The cation may be exchanged by treating a solution of the desiredprodrug anion with an appropriate cationic species to form an insolublesalt. The prodrug salt may be further purified by recrystallization ordrying in vacuo. Prodrug salt hydrates may be prepared, for example, bysalt formation, isolation, or recrystallization from aqueous solution.

Skilled artisans will recognize that in some instances, the active2,4-pyrimidinediamine compounds used as starting materials may includefunctional groups that require protection during synthesis. The exactidentity of any protecting group(s) used will depend upon the identityof the functional group being protected, and will be apparent to theseof skill in the art. Guidance for selecting appropriate protectinggroups, as well as synthetic strategies for their attachment andremoval, may be found, for example, in Greene & Wuts, Protective Groupsin Organic Synthesis, 3d Edition, John Wiley & Sons, Inc., New York(1999) and the references cited therein (hereinafter “Greene & Wuts”).

Inhibition of Fc Receptor Signal Cascades

Many of the prodrugs described herein, and in particular the prodrugsaccording to structural formulae (I) and (Ia), metabolize to active2,4-pyrimidinediamine compounds that inhibit Fc receptor signalingcascades that lead to, among other things, degranulation of cells. As aspecific example, these active compounds inhibit the FcεRI and/or FcγRIsignal cascades that lead to degranulation of immune cells such asneutrophil, eosinophil, mast and/or basophil cells. Both mast andbasophil cells play a central role in allergen-induced disorders,including, for example, allergic rhinitis and asthma. Upon exposureallergens, which may be, among other things, pollen or parasites,allergen-specific IgE antibodies are synthesized by B-cells activated byIL-4 (or IL-13) and other messengers to switch to IgE class specificantibody synthesis. These allergen-specific IgEs bind to the highaffinity FcεRI. Upon binding of antigen, the FcεR1-bound IgEs arecross-linked and the IgE receptor signal transduction pathway isactivated, which leads to degranulation of the cells and consequentrelease and/or synthesis of a host of chemical mediators, includinghistamine, proteases (e.g., tryptase and chymase), lipid mediators suchas leukotrienes (e.g., LTC4), platelet-activating factor (PAF) andprostaglandins (e.g., PGD2) and a series of cytokines, including TNF-α,IL-4, IL-13, IL-5, IL-6, IL-8, GMCSF, VEGF and TGF-β. The release and/orsynthesis of these mediators from mast and/or basophil cells accountsfor the early and late stage responses induced by allergens, and isdirectly linked to downstream events that lead to a sustainedinflammatory state.

The molecular events in the FcεRI signal transduction pathway that leadto release of preformed mediators via degranulation and release and/orsynthesis of other chemical mediators are well-known. The FcεRI is aheterotetrameric receptor composed of an IgE-binding alpha-subunit, abeta subunit, and two gamma subunits (gamma homodimer). Cross-linking ofFcεRI-bound IgE by multivalent binding agents (including, for exampleIgE-specific allergens or anti-IgE antibodies or fragments) induces therapid association and activation of the Src-related kinase Lyn. Lynphosphorylates immunoreceptor tyrosine-based activation motifs (ITAMS)on the intracellular beta and gamma subunits, which leads to therecruitment of additional Lyn to the beta subunit and Syk kinase to thegamma homodimer. These receptor-associated kinases, which are activatedby intra- and intermolecular phosphorylation, phosphorylate othercomponents of the pathway, such as the Btk kinase, LAT, andphospholipase C-gamma PLC-gamma). Activated PLC-gamma initiates pathwaysthat lead to protein kinase C activation and Ca²⁺ mobilization, both ofwhich are required for degranulation. FcεR1 cross-linking also activatesthe three major classes of mitogen activated protein (MAP) kinases, i.e.ERK1/2, JNK1/2, and p38. Activation of these pathways is important inthe transcriptional regulation of proinflammatory mediators, such asTNF-α and IL-6, as well as the lipid mediator leukotriene C4 (LTC4).

The FcγRI signaling cascade is believed to share some common elementswith the FceRI signaling cascade. Importantly, like FcεRI, the FcγRIincludes a gamma homodimer that is phosphorylated and recruits Syk, andlike FcεRI, activation of the FcγRI signaling cascade leads to, amongother things, degranulation. Other Fc receptors that share the gammahomodimer, and which can be regulated by the active2,4-pyrimidinediamine compounds include, but are not limited to, FcαRIand FcγRIII.

In vitro and cellular assays suitable for confirming the activity of aparticular 2,4-pyrimidinediamine compound are described in detail inU.S. application Ser. No. 10/355,543 filed Jan. 31, 2003(US2004/0029902A1), international application Serial No. PCT/US03/03022filed Jan. 31, 2003 (WO 03/063794), U.S. application Ser. No. 10/631,029filed Jul. 29, 2003 (US 2005/0028212), international application SerialNo. PCT/US03/24087 (WO2004/014382), U.S. application Ser. No. 10/903,263filed Jul. 30, 2004 (US2005/0234049), and international applicationSerial No. PCT/US2004/24716 (WO 2005/016893).

The ability of a particular prodrug to metabolize to an active2,4-pyrimidinediamine compound under the desired conditions of use canbe confirmed in in vitro and/or in vivo assays, as previously described.

Uses and Compositions

As previously discussed, the prodrugs and prodrug salts describedherein, such as those according to structural formulae (I), (Ia), (IX)and (IXa) metabolize when administered to animals and humans into activecompounds that inhibit Fc receptor signaling cascades, especially thoseFc receptors including a gamma homodimer, such as the FcεRI and/or FcγRIsignaling cascades, that lead to, among other things, the release and/orsynthesis of chemical mediators from cells, either via degranulation orother processes. As also discussed, the active compounds are also potentinhibitors of Syk kinase. As a consequence of these activities, prodrugsof these active compounds may be used in a variety of in vitro, in vivoand ex vivo contexts to regulate or inhibit Syk kinase, signalingcascades in which Syk kinase plays a role, Fc receptor signalingcascades, and the biological responses effected by such signalingcascades. For example, in one embodiment, the prodrugs and prodrug saltsmay be used to inhibit Syk kinase, either in vitro or in vivo, invirtually any cell type expressing Syk kinase. They may also be used toregulate signal transduction cascades in which Syk kinase plays a role.Such Syk-dependent signal transduction cascades include, but are notlimited to, the FcεRI, FcγRI, FcγRIII, BCR and integrin signaltransduction cascades. The prodrugs and prodrug salts may also be usedin vitro or in vivo to regulate, and in particular inhibit, cellular orbiological responses effected by such Syk-dependent signal transductioncascades. Such cellular or biological responses include, but are notlimited to, respiratory burst, cellular adhesion, cellulardegranulation, cell spreading, cell migration, cell aggregation,phagocytosis, cytokine synthesis and release, cell maturation and Ca²⁺flux. Importantly, the prodrugs and prodrug salts may be used to inhibitSyk kinase in vivo as a therapeutic approach towards the treatment orprevention of diseases mediated, either wholly or in part, by a Sykkinase activity. Non-limiting examples of Syk kinase mediated diseasesthat may be treated or prevented with the prodrugs and prodrug salts arethose discussed in more detail, below.

In another embodiment, the prodrugs and prodrug salts may be used toregulate or inhibit the Fc receptor signaling cascades and/or FcεRI-and/or FcγRI-mediated degranulation as a therapeutic approach towardsthe treatment or prevention of diseases characterized by, caused byand/or associated with the release or synthesis of chemical mediators ofsuch Fc receptor signaling cascades or degranulation. Such treatmentsmay be administered to animals in veterinary contexts or to humans.Diseases that are characterized by, caused by or associated with suchmediator release, synthesis or degranulation, and that can therefore betreated or prevented with the active compounds include, by way ofexample and not limitation, atopy or anaphylactic hypersensitivity orallergic reactions, allergies (e.g., allergic conjunctivitis, allergicrhinitis, atopic asthma, atopic dermatitis and food allergies), lowgrade scarring (e.g., of scleroderma, increased fibrosis, keloids,post-surgical scars, pulmonary fibrosis, vascular spasms, migraine,reperfusion injury and post myocardial infarction), diseases associatedwith tissue destruction (e.g., of COPD, cardiobronchitis and postmyocardial infarction), diseases associated with tissue inflammation(e.g., irritable bowel syndrome, spastic colon and inflammatory boweldisease), inflammation and scarring.

Recent studies have shown that activation of platelets by collagen ismediated through the same pathway used by immune receptors, with animmunoreceptor tyronsine kinase motif on the FcRγ playing a pivotal role(Watson & Gibbons, 1998, Immunol. Today 19:260-264), and also that FcRγplays a pivotal role in the generation of neointimal hyperplasiafollowing balloon injury in mice, most likely through collagen-inducedactivation of platelets and leukocyte recruitment (Konishi et al., 2002,Circulation 105:912-916). Thus, the prodrugs described herein can alsobe used to inhibit collagen-induced platelet activation and to treat orprevent diseases associated with or caused by such platelet activation,such as, for example, intimal hyperplasia and restenosis followingvascular injury.

In addition to the myriad diseases discussed above, cellular and animalempirical data confirm that the active 2,4-pyrimidinediamine compoundsdescribed in U.S. application Ser. No. 10/631,029 filed Jul. 29, 2003(US 2005/0028212), international application Serial No. PCT/US03/24087(WO2004/014382), U.S. application Ser. No. 10/903,263 filed Jul. 30,2004 (US2005/0234049), and international application Serial No.PCT/US2004/24716 (WO 2005/016893) are also useful for the treatment orprevention of autoimmune diseases, as well as the various symptomsassociated with such diseases. Thus, prodrugs and prodrug salts of theseactive compounds are useful for treating or preventing such diseasesand/or symptoms. The types of autoimmune diseases that may be treated orprevented with such prodrugs generally include those disorders involvingtissue injury that occurs as a result of a humoral and/or cell-mediatedresponse to immunogens or antigens of endogenous and/or exogenousorigin. Such diseases are frequently referred to as diseases involvingthe nonanaphylactic (i.e., Type II, Type III and/or Type IV)hypersensitivity reactions.

As discussed previously, Type I hypersensitivity reactions generallyresult from the release of pharmacologically active substances, such ashistamine, from mast and/or basophil cells following contact with aspecific exogenous antigen. As mentioned above, such Type I reactionsplay a role in numerous diseases, including allergic asthma, allergicrhinitis, etc.

Type II hypersensitivity reactions (also referred to as cytotoxic,cytolytic complement-dependent or cell-stimulating hypersensitivityreactions) result when immunoglobulins react with antigenic componentsof cells or tissue, or with an antigen or hapten that has becomeintimately coupled to cells or tissue. Diseases that are commonlyassociated with Type II hypersensitivity reactions include, but are notlimited, to autoimmune hemolytic anemia, erythroblastosis fetalis andGoodpasture's disease.

Type III hypersensitivity reactions, (also referred to as toxic complex,soluble complex, or immune complex hypersensitivity reactions) resultfrom the deposition of soluble circulating antigen-immunoglobulincomplexes in vessels or in tissues, with accompanying acute inflammatoryreactions at the site of immune complex deposition. Non-limitingexamples of prototypical Type III reaction diseases include the Arthusreaction, rheumatoid arthritis, serum sickness, systemic lupuserythematosis, certain types of glomerulonephritis, multiple sclerosisand bullous pemphingoid.

Type IV hypersensitivity reactions (frequently called cellular,cell-mediated, delayed, or tuberculin-type hypersensitivity reactions)are caused by sensitized T-lymphocytes which result from contact with aspecific antigen. Non-limiting examples of diseases cited as involvingType IV reactions are contact dermatitis and allograft rejection.

Autoimmune diseases associated with any of the above nonanaphylactichypersensitivity reactions may be treated or prevented with the prodrugsand prodrug salts according to structural formulae (I), (Ia), (IX) and(IXa). In particular, the methods may be used to treat or prevent thoseautoimmune diseases frequently characterized as single organ or singlecell-type autoimmune disorders including, but not limited to:Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmuneatrophic gastritis of pernicious anemia, autoimmune encephalomyelitis,autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia,sympathetic ophthalmia, myasthenia gravis, Graves' disease, primarybiliary cirrhosis, chronic aggressive hepatitis, ulcerative colitis andmembranous glomerulopathy, as well as those autoimmune diseasesfrequently characterized as involving systemic autoimmune disorder,which include but are not limited to: systemic lupus erythematosis(SLE), rheumatoid arthritis, Sjogren's syndrome, Reiter's syndrome,polymyositis-dermatomyositis, systemic sclerosis, polyarteritis nodosa,multiple sclerosis and bullous pemphigoid.

It will be appreciated by skilled artisans that many of the above-listedautoimmune diseases are associated with severe symptoms, theamelioration of which provides significant therapeutic benefit even ininstances where the underlying autoimmune disease may not beameliorated. Many of these symptoms, as well as their underlying diseasestates, result as a consequence of activating the FcγR signaling cascadein monocyte cells. As the prodrugs of structural formulae (I), (Ia),(IX) and (IXa) metabolize to 2,4-pyrimidinediamine compounds that arepotent inhibitors of such FcγR signaling in monocytes and other cells,the methods find use in the treatment and/or prevention of myriadadverse symptoms associated with the above-listed autoimmune diseases.

As a specific example, rheumatoid arthritis (RA) typically results inswelling, pain, loss of motion and tenderness of target jointsthroughout the body. RA is characterized by chronically inflamedsynovium that is densely crowded with lymphocytes. The synovialmembrane, which is typically one cell layer thick, becomes intenselycellular and assumes a form similar to lymphoid tissue, includingdendritic cells, T-, B- and NK cells, macrophages and clusters of plasmacells. This process, as well as a plethora of immunopathologicalmechanisms including the formation of antigen-immunoglobulin complexes,eventually result in destruction of the integrity of the joint,resulting in deformity, permanent loss of function and/or bone erosionat or near the joint. The methods may be used to treat or ameliorate anyone, several or all of these symptoms of RA. Thus, in the context of RA,the methods are considered to provide therapeutic benefit (discussedmore generally, infra) when a reduction or amelioration of any of thesymptoms commonly associated with RA is achieved, regardless of whetherthe treatment results in a concomitant treatment of the underlying RAand/or a reduction in the amount of circulating rheumatoid factor(“RF”).

The American College of Rheumatology (ACR) has developed criteria fordefining improvement and clinical remission in RA. Once such parameter,the ACR20 (ACR criteria for 20% clinical improvement), requires a 20%improvement in the tender and swollen joint count, as well as a 20%improvement in 3 of the following 5 parameters: patient's globalassessment, physician's global assessment, patient's assessment of pain,degree of disability, and level of acute phase reactant. These criteriahave been expanded for 50% and 70% improvement in ACR50 and ACR70,respectively. Other criteria includes Paulu's criteria and radiographicprogression (e.g. Sharp score).

In some embodiments, therapeutic benefit in patients suffering from RAis achieved when the patient exhibits an ARC20. In specific embodiments,ARCs of ARC50 or even ARC70 may be achieved.

Systemic lupus erythematosis (“SLE”) is typically associated withsymptoms such as fever, joint pain (arthralgias), arthritis, andserositis (pleurisy or pericarditis). In the context of SLE, the methodsare considered to provide therapeutic benefit when a reduction oramelioration of any of the symptoms commonly associated with SLE areachieved, regardless of whether the treatment results in a concomitanttreatment of the underlying SLE.

Multiple sclerosis (“MS”) cripples the patient by disturbing visualacuity; stimulating double vision; disturbing motor functions affectingwalking and use of the hands; producing bowel and bladder incontinence;spasticity; and sensory deficits (touch, pain and temperaturesensitivity). In the context of MS, the methods are considered toprovide therapeutic benefit when an improvement or a reduction in theprogression of any one or more of the crippling effects commonlyassociated with MS is achieved, regardless of whether the treatmentresults in a concomitant treatment of the underlying MS.

When used to treat or prevent such diseases, the prodrugs and prodrugsalts described herein may be administered singly, as mixtures of one ormore prodrugs and prodrug salts or in mixture or combination with otheragents useful for treating such diseases and/or the symptoms associatedwith such diseases. The prodrugs and prodrug salts may also beadministered in mixture or in combination with agents useful to treatother disorders or maladies, such as steroids, membrane stabilizers, 5LOinhibitors, leukotriene synthesis and receptor inhibitors, inhibitors ofIgE isotype switching or IgE synthesis, IgG isotype switching or IgGsynthesis, β-agonists, tryptase inhibitors, aspirin, COX inhibitors,methotrexate, anti-TNF drugs, retuxin, PD4 inhibitors, p38 inhibitors,PDE4 inhibitors, and antihistamines, to name a few. The prodrugs andprodrug salts may be administered in the form of compounds per se, or aspharmaceutical compositions comprising a prodrug.

Another aspect of the invention relates to pharmaceutical compositionsof the prodrugs, prodrug salts, prodrug salt hydrates, and crystalsthereof as described herein. Accordingly, one embodiment of theinvention provides a pharmaceutical composition comprising a prodrug,prodrug salt, prodrug salt hydrate or crystal, and apharmaceutically-acceptable carrier, excipient and/or diluent. Theprodrug, prodrug salt, prodrug salt hydrate or crystal may be made, forexample, by any of the methods described herein. Preferably, the moleratio of the prodrug, prodrug salt, prodrug salt hydrate, or crystal tothe corresponding drug compound is greater than about 18:1. For example,when the pharmaceutical composition includes the prodrug salt hydrate

the pharmaceutical composition has a mole ratio of prodrug salt hydrateto the corresponding drug compound

of at least about 10:1. Preferably, the ratio is at least about 18:1.

Pharmaceutical compositions comprising the prodrug(s) and prodrugsalt(s) may be manufactured by means of conventional mixing, dissolving,granulating, dragee-making levigating, emulsifying, encapsulating,entrapping or lyophilization processes. The compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the prodrugs into preparations which can beused pharmaceutically.

The prodrug may be formulated in the pharmaceutical composition per se,or in the form of a hydrate, solvate, N-oxide or pharmaceuticallyacceptable salt, as previously described. Typically, such salts are moresoluble in aqueous solutions than the corresponding free acids andbases, but salts having lower solubility than the corresponding freeacids and bases may also be formed.

Pharmaceutical compositions may take a form suitable for virtually anymode of administration, including, for example, topical, ocular, oral,buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc.,or a form suitable for administration by inhalation or insufflation.

For topical administration, the prodrug(s) and prodrug salt(s) may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives.

Alternatively, the injectable formulation may be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, etc., before use.To this end, the active compound(s) may be dried by any art-knowntechnique, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the pharmaceutical compositions may take theform of, for example, lozenges, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets may be coated by methods well known in theart with, for example, sugars, films or enteric coatings. Phosphateprodrugs in which the progroup(s) is of the formula—(CR^(d)R^(d))_(y)—O—P(O)(OH)₂, and certain phosphate prodrug salts inwhich the progroup(s) is of the formula—(CR^(d)R^(d))_(y)—O—P(O)(O⁻)₂[M⁺]₂ or—(CR^(d)R^(d))_(y)—O—P(O)(OH)(O⁻)[M⁺], where each R^(d) is,independently of the others, selected from hydrogen and lower alkyl andy is 1 or 2 and that exhibit a water-solubility in the range of about0.1 to 1000 mg/ml at physiological pH are especially suited for oraladministration via tablets and capsules. When administered toSprague-Dawley rats orally from capsules, prodrug Compound 4 exhibits abioavailability of drug Compound 1 of about 30% (see FIG. 5), withabsorption being nearly identical to that of active drug Compound 1 (seeFIG. 6). Other phosphate prodrugs and prodrug salts havingwater-solubility properties similar to those of prodrug Compound 4 areexpected to exhibit similar pharmacokinetic properties.

A specific exemplary tablet formulation for prodrug Compound 4 (as wellas other phosphate-containing prodrugs) contains about 50-400 mg prodrugcompound (or a salt thereof), about 0.05 to 0.5 wt % colloidal silicondioxide, about 0.5 to 5.0 wt % croscarmellose sodium, about 0.25 to 5.0wt % magnesium stearate and about 20 to 80 wt % microcrystallinecellulose. If desired, the tablets can be coated with a film, such as ahypromellose film carboxymethyl cellulose or fructose, which canoptionally contain coloring agents, such as for example FD&C blue #1,PD&C green #3, FD&C yellow #6 and titanium dioxide.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, CREMOPHORE™ or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, preservatives, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the prodrug or prodrug salt, as is well known.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For rectal and vaginal routes of administration, the prodrug(s) orprodrug salt(s) may be formulated as solutions (for retention enemas)suppositories or ointments containing conventional suppository basessuch as cocoa butter or other glycerides.

For nasal administration or administration by inhalation orinsufflation, the prodrug(s) or prodrug salt(s) can be convenientlydelivered in the form of an aerosol spray from pressurized packs or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or othersuitable gas. In the case of a pressurized aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount. Capsulesand cartridges for use in an inhaler or insufflator (for examplecapsules and cartridges comprised of gelatin) may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

For ocular administration, the prodrug(s) or prodrug salt(s) may beformulated as a solution, emulsion, suspension, etc. suitable foradministration to the eye. A variety of vehicles suitable foradministering compounds to the eye are known in the art. Specificnon-limiting examples are described in U.S. Pat. No. 6,261,547; U.S.Pat. No. 6,197,934; U.S. Pat. No. 6,056,950; U.S. Pat. No. 5,800,807;U.S. Pat. No. 5,776,445; U.S. Pat. No. 5,698,219; U.S. Pat. No.5,521,222; U.S. Pat. No. 5,403,841; U.S. Pat. No. 5,077,033; U.S. Pat.No. 4,882,150; and U.S. Pat. No. 4,738,851.

For prolonged delivery, the prodrug(s) or prodrug salt(s) can beformulated as a depot preparation for administration by implantation orintramuscular injection. The prodrug(s) or prodrug salt(s) may beformulated with suitable polymeric or hydrophobic materials (e.g., as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, e.g., as a sparingly soluble salt. Alternatively,transdermal delivery systems manufactured as an adhesive disc or patchwhich slowly releases the prodrug(s) or prodrug salt(s) for percutaneousabsorption may be used. To this end, permeation enhancers may be used tofacilitate transdermal penetration of the prodrug(s) or prodrug salt(s).Suitable transdermal patches are described in for example, U.S. Pat. No.5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat.No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S.Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977;U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No.4,921,475.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well-known examples of delivery vehiclesthat may be used to deliver prodrug(s) or prodrug salt(s). Certainorganic solvents such as dimethylsulfoxide (DMSO) may also be employed,although usually at the cost of greater toxicity.

The pharmaceutical compositions may, if desired, be presented in a packor dispenser device which may contain one or more unit dosage formscontaining the prodrug(s) or prodrug salt(s). The pack may, for example,comprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.

Effective Dosages

The prodrug(s) or prodrug salt(s) described herein, or compositionsthereof, will generally be used in an amount effective to achieve theintended result, for example in an amount effective to treat or preventthe particular disease being treated. The prodrug(s) or prodrug salt(s)may be administered therapeutically to achieve therapeutic benefit orprophylactically to achieve prophylactic benefit. By therapeutic benefitis meant eradication or amelioration of the underlying disorder beingtreated and/or eradication or amelioration of one or more of thesymptoms associated with the underlying disorder such that the patientreports an improvement in feeling or condition, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forexample, administration of a compound to a patient suffering from anallergy provides therapeutic benefit not only when the underlyingallergic response is eradicated or ameliorated, but also when thepatient reports a decrease in the severity or duration of the symptomsassociated with the allergy following exposure to the allergen. Asanother example, therapeutic benefit in the context of asthma includesan improvement in respiration following the onset of an asthmaticattack, or a reduction in the frequency or severity of asthmaticepisodes. Therapeutic benefit in the context of RA also includes theACR20, or ACR50 or ACR70, as previously described. Therapeutic benefitalso generally includes halting or slowing the progression of thedisease, regardless of whether improvement is realized.

For prophylactic administration, the prodrug(s) or prodrug salt(s) maybe administered to a patient at risk of developing one of the previouslydescribed diseases. For example, if it is unknown whether a patient isallergic to a particular drug, the prodrug(s) or prodrug salt(s) may beadministered prior to administration of the drug to avoid or amelioratean allergic response to the drug. Alternatively, prophylacticadministration may be applied to avoid the onset of symptoms in apatient diagnosed with the underlying disorder. For example, theprodrug(s) or prodrug salt(s) may be administered to an allergy suffererprior to expected exposure to the allergen. Prodrug(s) or prodrugsalt(s) may also be administered prophylactically to healthy individualswho are repeatedly exposed to agents known to one of the above-describedmaladies to prevent the onset of the disorder. For example, prodrug(s)or prodrug salt(s) may be administered to a healthy individual who isrepeatedly exposed to an allergen known to induce allergies, such aslatex, in an effort to prevent the individual from developing anallergy. Alternatively, prodrug(s) or prodrug salt(s) may beadministered to a patient suffering from asthma prior to partaking inactivities which trigger asthma attacks to lessen the severity of, oravoid altogether, an asthmatic episode.

The amount of prodrug(s) or prodrug salt(s) administered will dependupon a variety of factors, including, for example, the particularindication being treated, the mode of administration, whether thedesired benefit is prophylactic or therapeutic, the severity of theindication being treated and the age and weight of the patient, thebioavailability of the particular prodrug(s) or prodrug salt(s) theconversation rate and efficiency into active drug compound under theselected route of administration, etc. Determination of an effectivedosage of prodrug(s) or prodrug salt(s) for a particular use and mode ofadministration is well within the capabilities of those skilled in theart.

Effective dosages may be estimated initially from in vitro activity andmetabolism assays. For example, an initial dosage of prodrug or prodrugsalt for use in animals may be formulated to achieve a circulating bloodor serum concentration of the metabolite active compound that is at orabove an IC₅₀ of the particular compound as measured in as in vitroassay, such as the in vitro CHMC or BMVIC and other in vitro assaysdescribed in U.S. application Ser. No. 10/355,543 filed Jan. 31, 2003(US2004/0029902A1), international application Serial No. PCT/US03/03022filed Jan. 31, 2003 (WO 03/063794), U.S. application Ser. No. 10/631,029filed Jul. 29, 2003 (US 2005/0028212), international application SerialNo. PCT/US03/24087 (WO2004/014382), U.S. application Ser. No. 10/903,263filed Jul. 30, 2004 (US2005/0234049), and international applicationSerial No. PCT/US2004/24716 (WO 2005/016893). Calculating dosages toachieve such circulating blood or serum concentrations taking intoaccount the bioavailability of the particular prodrug via the desiredroute of administration is well within the capabilities of skilledartisans. For guidance, the reader is referred to Fingl & Woodbury,“General Principles,” In: Goodman and Gilman's The Pharmaceutical Basisof Therapeutics, Chapter 1, pp. 1-46, latest edition, Pagamonon Press,and the references cited therein.

Initial dosages of prodrug or prodrug salt can also be estimated from invivo data, such as animal models. Animal models useful for testing theefficacy of the active metabolites to treat or prevent the variousdiseases described above are well-known in the art. Suitable animalmodels of hypersensitivity or allergic reactions are described inFoster, 1995, Allergy 50(21Suppl):6-9, discussion 34-38 and Tumas etal., 2001, J. Allergy Clin. Immunol. 107(6):1025-1033. Suitable animalmodels of allergic rhinitis are described in Szelenyi et al., 2000,Arzneimittelforschung 50(11):1037-42; Kawaguchi et al., 1994, Clin. Exp.Allergy 24(3):238-244 and Sugimoto et al., 2000, Immunopharmacology48(1):1-7. Suitable animal models of allergic conjunctivitis aredescribed in Carreras et al., 1993, Br. J. Ophthalmol. 77(8):509-514;Saiga et al., 1992, Ophthalmic Res. 24(1):45-50; and Kunert et al.,2001, Invest. Ophthalmol. Vis. Sci. 42(11):2483-2489. Suitable animalmodels of systemic mastocytosis are described in O'Keefe et al., 1987,J. Vet. Intern. Med. 1(2):75-80 and Bean-Knudsen et al., 1989, Vet.Pathol. 26(1):90-92. Suitable animal models of hyper IgE syndrome aredescribed in Claman et al., 1990, Clin. Immunol. Immunopathol.56(1):46-53. Suitable animal models of B-cell lymphoma are described inHough et al., 1998, Proc. Natl. Acad. Sci. USA 95:13853-13858 and Hakimet al., 1996, J. Immunol. 157(12):5503-5511. Suitable animal models ofatopic disorders such as atopic dermatitis, atopic eczema and atopicasthma are described in Chan et al., 2001, J. Invest. Dermatol.117(4):977-983 and Suto et al., 1999, Int. Arch. Allergy Immunol.120(Suppl 1):70-75. Animal models suitable for testing thebioavailability and/or metabolism of prodrugs into active metabolitesare also well-known. Ordinarily skilled artisans can routinely adaptsuch information to determine dosages of particular prodrugs and prodrugsalts suitable for human administration. Additional suitable animalmodels are described in the Examples section.

Dosage amounts will typically be in the range of from about 0.0001mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, butmay be higher or lower, depending upon, among other factors, theactivity of the active metabolite compound, the bioavailability of theprodrug or prodrug salt, its metabolism kinetics and otherpharmacokinetic properties, the mode of administration and various otherfactors, discussed above. Dosage amount and interval may be adjustedindividually to provide plasma levels of the prodrug(s), prodrug salt(s)and/or active metabolite compound(s) which are sufficient to maintaintherapeutic or prophylactic effect. For example, the prodrugs may beadministered once per week, several times per week (e.g., every otherday), once per day or multiple times per day, depending upon, amongother things, the mode of administration, the specific indication beingtreated and the judgment of the prescribing physician. In cases of localadministration or selective uptake, such as local topicaladministration, the effective local concentration of prodrug(s), prodrugsalt(s) and/or active metabolite compound(s) may not be related toplasma concentration. Skilled artisans will be able to optimizeeffective local dosages without undue experimentation.

Preferably, the prodrugs will metabolize into active compound(s) thatwill provide therapeutic or prophylactic benefit without causingsubstantial toxicity. Toxicity of the active and other metabolites, aswell as the unmetabolized prodrug may be determined using standardpharmaceutical procedures. The dose ratio between toxic and therapeutic(or prophylactic) effect is the therapeutic index. Prodrug(s) andprodrug salt(s) that exhibit high therapeutic indices are preferred.

The inventions having been described, the following examples are offeredby way of illustration and not limitation.

EXAMPLES 1. Synthesis of Prodrug Compound 4 1.1N4-(2,2-dimethyl-4-[(di-tert-butylphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 3)

N4-(2,2-dimethyl-3-oxo-4H-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(1, 1.0 g, 2.12 mmol), Cs₂CO₃ (1.0 g, 3.07 mmol) and di-tert-butylchloromethyl phosphate (2, 0.67 g, 2.59 mmol) in acetone (20 mL) wasstirred at room temperature under nitrogen atmosphere. Progress of thereaction was monitored by LC/MS. Crude reaction mixture displayed threeproduct peaks with close retention times with M⁺+H 693 (minor-1), 693(major; 3) and 477 (minor-2) besides starting material (Compound 1).Upon stirring the contents for 4 days (70% consumption), the reactionmixture was concentrated and diluted with water. The resultant paleyellow precipitate formed was collected by filtration and dried. Thecrude solid was purified by silica gel (pretreated with 10% NEt3/CH2Cl2followed by eluting with hexanes) column chromatography by gradientelution with 70% EtOAc/hexanes-100% EtOAc). The fractions containingCompound 1 and M++H 693 were collected and concentrated. The resultingcrude white solid was subjected to repurification in the similar manneras described previously but by eluting with 30%-50%-75%-100%EtOAc/hexanes. The major product peak with M++H 693 was collected as awhite solid (270 mg, 18%) and was characterized asN4-(2,2-dimethyl-4-[(di-tert-butylphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 3). 1H NMR (DMSO-d6): δ 9.21 (s, 1H), 9.17 (s, 1H), 8.16 (d,1H, J=2.6 Hz), 7.76 (d, 1H, J=8.5 Hz), 7.44 (d, 1H, J=8.5 Hz), 7.02 (s,2H), 5.78 (d, 1H, J3PH=6.1 Hz), 3.64 (s, 6H), 3.58 (s, 3H), 1.45 (s,6H), 1.33 (s, 9H). LCMS: ret. time: 14.70 min.; purity: 95%; MS (m/e):693 (MH+). 31P NMR (DMSO-d6): −11.36.

1.2. N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 4)

Trifluoroacetic acid (1.5 mL) was added dropwise as a neat for 5 min toN4-(2,2-dimethyl-4-[(di-tert-butylphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 3, 120 mg, 0.173 mmol) dissolved in CH₂Cl₂ (10 mL) at 0° C.under nitrogen atmosphere. The contents were allowed to stir for 1.5 h.Progress of the reaction mixture was monitored by LC/MS. After completeconsumption of the starting material, reaction mixture was concentrated,dried and triturated with ether. The ethereal layer was decanted anddried to provide the crude solid. LC/MS analysis of the crude displayedthree peaks with M⁺+H 581, 471 and 501. The peak corresponding to M⁺+H581 was collected by preparative HPLC chromatographic purification. Thefractions were lyophilised and dried to provide 53 mg (52%) of off whitefluffy solid and characterized as N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 4). ¹H NMR (DMSO-d6): δ 9.21 (br s, 2H), 8.16 (d, 1H, J=2.6Hz), 7.93 (d, 1H, J=8.5 Hz), 7.39 (d, 1H, J=8.5 Hz), 7.05 (s, 2H), 5.79(d, 1H, J³ _(PH)=6.6 Hz), 3.67 (s, 6H), 3.59 (s, 3H), 1.44 (s, 6H).LCMS: ret. time: 8.52 min.; purity: 95%; MS (m/e): 581 (MH⁺). ³¹P NMR(DMSO-d6): −2.17.

2. Alternative Synthesis of Prodrug Compound 4

An alternative method of synthesizing prodrug Compound 4 whichalleviates the need for column chromatography and HPLC purification isprovided below.

2.1 Synthesis of N4-(2,2-dimethyl-4-[(di-tert-butylphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 3)

N4-(2,2-dimethyl-3-oxo-4H-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 1, 19.73 g, 41.97 mmol), Cs₂CO₃ (15.04 g, 46.16 mmol) anddi-tert-butyl chloromethyl phosphate (13.0 g, 50.38 mmol) in DMF (100mL) was stirred at room temperature under nitrogen atmosphere. Progressof the reaction was monitored by in process LC/MS. Crude reactionmixture displayed two product peaks (ratio 1:6.5) with close retentiontimes displaying M⁺+H 693 (minor) and 693 (major) besides startingmaterial (Compound 1). Initial yellow reaction mixture turned to olivegreen as the reaction progressed. Workup was carried out as follows

1). Upon stirring the contents for 30 h (92% consumption), reactionmixture was poured onto ice-water (400 mL) and stirred the contents byadding brine solution (200 mL). Fine yellow tan solid formed wasfiltered, washed with water and dried overnight.

2). The solid (35 g) was dissolved in MTBE (500 mL) and washed withwater (400 mL). Aqueous layer was extracted with MTBE (2×350 mL) tillthe absence of UV on TLC. Combined organic layers were dried overanhydrous Na₂SO₄ and decanted.

Note: step 2 can be done directly, however, DMF extraction back intosolution leads to difficulty in the crystallization step.

3). The dark red clear solution was subjected to 10 g of activatedcharcoal treatment, heated to boil and filtered.

4). The dark red clear solution was concentrated by normal heating to400 mL of its volume and left for crystallization. The solidcrystallized as granules was filtered, crushed the granules to powder,washed with MTBE (400 mL) and dried under high vacuum. See step 7 forthe workup of mother liquor. Weight of the solid: 17 g; purity: 90%(Compound 3), 6.26% (Compound 1), 1.8% (minor M+693).

5). At this stage solid was taken in 500 ml of ethyl ether and heated toboil. Cooled and filtered to remove undissolved material. Filtrate wasconcentrated.

6). Above concentrate was subjected to crystallization in MTBE (300 mL).The white solid formed was filtered, washed with MTBE (100 mL) and driedunder high vacuum to provide the desiredN4-(2,2-dimethyl-4-[(di-tert-butylphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 3) in 97% purity. ¹H NMR (DMSO-d6): δ 9.21 (s, 1H), 9.17 (s,1H), 8.16 (d, 1H, J=2.6 Hz), 7.76 (d, 1H, J=8.5 Hz), 7.44 (d, 1H, J=8.5Hz), 7.02 (s, 2H), 5.78 (d, 1H, J³PH=6.1 Hz), 3.64 (s, 6H), 3.58 (s,3H), 1.45 (s, 6H), 1.33 (s, 9H). LCMS: ret. time: 14.70 min.; purity:95%; MS (m/e): 693 (MH⁺). ³¹P NMR (DMSO-d6): −11.36. Weight of thesolid: 15.64 g (yield: 55%); purity: 97% (Compound 3), 3% (Compound 1).

7). The mother liquor was concentrated and steps 5 and 6 were repeatedto provide Compound 3.

2.2. Synthesis of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 4)

N4-(2,2-dimethyl-4-[(di-tert-butylphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 3); (15.0 g, 21.67 mmol) dissolved in AcOH:H₂O (225 mL, 4:1)was heated at 65° C. (oil bath temp). The progress of the reaction wasmonitored by in process LC/MS. The reaction mixture transformed to fainttan white solid after 1 h of heating. At this point most of Compound 3converted to mono des t-butyl product. After 3 h of heating, consumptionof SM and complete conversion of intermediate (mono des t-butylated) toproduct was observed.

Reaction mixture was cooled, poured onto ice-water (200 mL), stirred for20 min and filtered. The clear white filter cake was washed with water(600 ml) and acetone (200 mL) successively, dried for 2 h followed bydrying under high vacuum over P₂O₅ in a desiccator. Weight of the solid:12.70 g; purity: 97% (Compound 3) and 3% (Compound 1)¹H NMR indicatedacetic acid presence (1:1)

To remove acetic acid, the solid was taken in acetonitrile (300 mL) andconcentrated by rotovap vacuum. This process was repeated 2 times withacetonitrile and toluene (3×300 mL). The solid obtained was dried underhigh vacuum at 50° C.

Finally, the solid was taken in acetone (400 mL), filtered and dried toprovide solid N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 4). ¹H NMR (DMSO-d6): δ 9.21 (br s, 2H), 8.16 (d, 1H, J=2.6Hz), 7.93 (d, 1H, J=8.5 Hz), 7.39 (d, 1H, J=8.5 Hz), 7.05 (s, 2H), 5.79(d, 1H, J³ _(PH)=6.6 Hz), 3.67 (s, 6H), 3.59 (s, 3H), 1.44 (s, 6H).LCMS: ret. time: 8.52 min.; purity: 95%; MS (m/e): 581 (MH⁺). ³¹P NMR(DMSO-d6): −2.17.

3. Synthesis of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminemono calcium salt (Prodrug Salt 6)

Aqueous (10 mL) NaHCO₃ (0.17 g, 2.02 mmol) solution was added dropwiseto a suspension of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(0.5 g, 0.86 mmol) in water (5 mL) at room temperature while stirringthe contents. The clear solution formed was treated with aqueous (10 mL)CaCl₂ (0.11 g in 10 mL water, 0.99 mmol) n a dropwise manner at roomtemperature. The addition resulted in the precipitation of a white solidfrom reaction mixture. Upon completion of addition, the contents werestirred for a period of 30 min, filtered, washed with water (40 mL) anddried. The clear white solid was taken in water (30 mL) and heated on astir plate to boil. The solution was cooled, filtered and dried. Thewhite solid collected and further dried under high vacuo at 80° C. for32 h to provide 0.41 g (83%) of solid N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminemono calcium salt (Prodrug Salt 6).

Ca(OAc)₂ may also used in place of CaCl₂ in this preparation.

4. Synthesis of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminedisodium salt hexahydrate and monosodium salt hydrate

A round-bottomed flask was charged with 10.00 gN4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 4) and 140 mL water into a round bottom flask to form a slurryhaving a pH between 3.6 and 3.7. The pH was adjusted to in the range of9.3 to 10.3 by addition of 1 M aqueous NaOH, initially forming a turbidsolution, which returned to a suspension upon prolonged stirring. Themixture was heated at reflux, then the turbid solution was hot filteredthrough filter paper. The solid collected in the filter paper was rinsedwith 10 mL hot water. Isopropanol (75 mL) was added to the filtrate,yielding a clear solution, which was allowed to cool to room temperatureover about 1.5 hours with stirring, during which time a solidprecipitated. The precipitate was collected by filtration, rinsed with47 mL isopropanol, and taken up in 73 mL acetone to form a slurry, whichwas stirred for 1.5 hours at room temperature. The solid was againcollected by filtration and rinsed with 18 mL acetone, then dried atabout 40° C. under vacuum until substantially all isopropanol andacetone was removed (i.e., below 0.5 wt % each). The product was exposedto air at about 40% relative humidity and room temperature until thewater content stabilized at about 15% by Karl Fisher titration, yielding8.18 g of the title compound. ¹H NMR (D₂O): δ 7.67 (d, 1H, J=3.8 Hz),7.49 (d, 1H, J=8.8 Hz), 6.87 (d, 1H, J=8.8 Hz), 6.50 (s, 2H), 5.52 (d,1H, J³PH=2.0 Hz), 3.53 (s, 3H), 3.47 (s, 6H), 1.32 (s, 6H). ³¹P NMR(D₂O): 2.75. The prodrug salt hydrate was obtained as a pure-white,highly crystalline material. Microscopic investigation indicated thatthe crystallites are plate-like with a particle size of less than 10 μm.Polarized light microscopy revealed birefringence corroborating thecrystalline nature of the hydrate.

The monosodium salt can be prepared from N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamineand sodium hydroxide by a proper pH control; pH of 5-5.5 results inpredominantly the formation of monosodium salt.

5. Preparation of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminedipotassium salt

A suspension of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine,acetic acid complex (1.0 g, 1.56 mmol) in water (15 mL) was heated at70° C. (oil bath temp) for 10 min (pH=2.9). To the above stirringsuspension, aqueous KOH (2.1 M, 1.5 ml) was added dropwise and the pHwas observed as 5.9. At this point 2.5 M aqueous KOH was added dropwisewhile monitoring the pH. When the pH reached to 10.5 (after 0.95 mL),addition was stopped and the clear solution stirred at the sametemperature for 15 min. The warm solution was filtered into a conicalflask and washed the filter paper with water to a combined volume of 45mL. The filtrate was transferred onto a hot plate and isopropanol (175mL) was added portionwise to the hot solution, until the turbiditypersisted upon heating. Water was then added dropwise until the solutionwas just clear at its boiling point. The conical flask was removed fromthe hot plate and allowed to cool to room temperature. A crystallinesolid formed, which was collected by suction filtration, washed withminimum amount of isopropanol and dried for 30 min. The resultant whitesolid was dried under vacuum overnight at 70° C., yieldingN4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminedipotassium salt, (0.95 g, 1.44 mmol, 92%, 99% pure) ¹H NMR (D₂O): δ7.68 (d, 1H, J=3.8 Hz), 7.49 (d, 1H, J=8.8 Hz), 6.87 (d, 1H, J=8.8 Hz),6.51 (s, 2H), 5.52 (d, 1H, J³ _(PH)=2.0 Hz), 3.54 (s, 3H), 3.48 (s, 6H),1.32 (s, 6H). ³¹P NMR (D₂O): 2.7

6. Preparation of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminedi-L-Arginine Salt

A suspension of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(0.25 g, 0.43 mmol) and L-arginine (0.15 g, 0.86 mmol) in EtOH (15 mL)was heated at 90° C. (oil bath temp) for 10 min. Water (7.5 mL) wasadded dropwise to the hot stirring suspension until it became a clearsolution. After 1 h of heating with stirring, the hot solution wasfiltered into an Erlenmeyer flask. The filtrate in the Erlenmeyer flaskwas brought to boiling on a hot plate, the allowed to cool to roomtemperature. A solid formed, which was collected by gravity filtrationand dried under vacuum overnight at 80° C. to form the subject prodrugsalt hydrate (0.28 g, 0.3 mmol, 69%). ¹H NMR (D₂O): δ □7.64 (d, 1H,J=3.5 Hz), 7.42 (d, 1H, J=8.8 Hz), 6.80 (d, 1H, J=8.8 Hz), 6.45 (s, 2H),5.53 (d, 1H, J³ _(PH)=2.8 Hz), 3.57 (t, 2H, J=6.0 Hz), 3.51 (s, 3H),3.44 (s, 6H), 3.01 (t, 4H, J=6.5 Hz), 1.74-1.69 (m, 4H), 1.55-1.46 (m,4H), 1.30 (s, 6H). ³¹P NMR (D₂O): 2.56.

7. Preparation of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminedi-L-lysine Salt

A suspension of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(0.25 g, 0.43 mmol) and L-lysine (0.125 g, 0.86 mmol) in EtOH (15 mL)was heated at 90° C. (oil bath temp) for 10 min. Water (4.5 mL) wasadded dropwise to the hot stirring suspension until it formed a clearsolution. After 1 h of heating and stirring, the reaction mixturefiltered, cooled and concentrated under vacuum. Precipitation of thecrude concentrate was observed upon addition of EtOH (5 mL). Theresultant solid was stirred overnight at room temperature in t-BuOMeafter concentration of the mixture. The white solid was collected bygravity filtration and dried under vacuum overnight at 80° C. (0.32 g,83%). ¹H NMR (D₂O): δ 7.67 (d, 1H, J=3.8 Hz), 7.47 (d, 1H, J=8.8 Hz),6.84 (d, 1H, J=8.8 Hz), 6.48 (s, 2H), 5.54 (d, 1H, J³ _(PH)=3.5 Hz),3.57 (t, 2H, J=6.0 Hz), 3.51 (s, 3H), 3.44 (s, 6H), 2.86 (t, 4H, J=6.7Hz), 1.77-1.70 (m, 4H), 1.62-1.52 (app q, 4H, J 6.3 Hz), 1.38-1.26 (m,10H). ³¹P NMR (D₂O): 2.59.

8. Synthesis of N4-(2, 2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminemono magnesium salt

A suspension of N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminedipotassium salt (0.5 g, 0.76 mmol) in 10 mL water was placed ontopreheated oil bath at 80° C. and stirred till the suspension formed aclear solution. The hot solution was filtered, and the filter paper waswashed with another 10 mL of water. The clear filtrate was heated whilestirring at 80° C. MgCl₂ (0.076 g, 0.8 mmol) was dissolved in water (10mL), filtered into a flask through a filter column (rinsing with 10 mLwater), and heated at 90° C. (pH=7.52). The preheated dipotassium saltsolution was added dropwise to the above MgCl₂ solution for 15 min whilestirring the contents. The initial white frothing suspension formedslowly turned to clear white solid upon heating the contents at 80° C.for 1.5 h (pH=6.3-6.7). The solid was collected by suction filtrationand washed with water until there was no chloride ion was detected(AgNO₃ test). The solid was suction dried for 2 h, then by vacuum driedat 70° C. overnight to provide N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediaminemono magnesium salt (0.43 mg, 93%).

9. Synthesis of Prodrug Compound 8

N4-(2,2-dimethyl-4-[(di-tert-butylphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(prepared as described above) (0.2 g, 0.29 mmol) was added to a mixtureof MeOH (5 mL) and Et₂O (5 mL). 2N aq. NaOH (0.023 g, 0.58 mmol) wasadded at once while stirring the contents at room temperature. Progressof the reaction was monitored by LC/MS. After 8 h of stirring, the solidprecipitated was filtered and dried to provideN4-(2,2-dimethyl-4-methoxymethyl-3-oxo-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 8) as a white solid (0.11 g, 74%). ¹H NMR (DMSO-d6): δ 9.47(s, 1H), 9.15 (s, 1H), 8.16 (d, 1H, J=3.8 Hz), 7.87 (d, 1H, J=8.5 Hz),7.37 (d, 1H, J=8.5 Hz), 7.03 (s, 2H), 5.40 (s, 2H), 3.66 (s, 6H), 3.59(s, 3H), 3.27 (s, 3H), 1.44 (s, 6H). LCMS: ret. time: 12.88 min.;purity: 92%; MS (m/e): 515 (MI-t).

10. The Active 2,4-Pyrimidinediamine Compounds Are Tolerated In Animals

The ability of numerous biologically active 2,4-pyrimidinediaminecompounds to exert their activity at doses below those exhibitingtoxicity in animals has been demonstrated previously (see, e.g., U.S.application Ser. No. 10/355,543 filed Jan. 31, 2003 (US2004/0029902A1),international application Serial No. PCT/US03/03022 filed Jan. 31, 2003(WO 03/063794), U.S. application Ser. No. 10/631,029 filed Jul. 29, 2003(US 2005/0028212), international application Serial No. PCT/US03/24087(WO2004/014382), U.S. application Ser. No. 10/903,263 filed Jul. 30,2004 (US2005/0234049), and international application Serial No.PCT/US2004/24716 (WO 2005/016893).

The safety pharmacology of active Compound 1 has been studied in a corebattery of studies (respiratory, CNS, cardiovascular, and HERG). Aslight reduction in heart rate and increase in RR interval was noted at50 mg/kg in the cardiovascular study and a slight effect on a fewbehavioral parameters at 50 mg/kg was also noted in the CNS (Irwin)study. Otherwise the safety pharmacology studies determined thatCompound 1 was well tolerated. GLP toxicology studies included negativemutagenicity and clastogenicity studies (Ames, chromosomal aberration,and mouse micronucleus). In 28-day toxicity studies in rats and monkeys,higher doses had evidence of a reversible effect on hematology, livertransaminase (mild effect in the rat only), spleen and thymus size (ratonly) and bone marrow cellularity (rat and monkey). Immunophenotyping inthe rat study revealed a significant decrease in the percentage of CD3+cells in high dose rats while a significant increase in CD45RA+ cellswas noted following recovery. Histopathology was noteworthy only formild reductions in marrow cellularity at high doses. There was noevidence for untoward effects on humoral immunity in the anti-KLHantibody assessment. The No Observed Adverse Effect Level (NOAEL) is10-30 mg/kg/day for rats and 100 mg/kg/day for monkeys.

11. Drug Compound 1 is Biologically Active in In Vitro Assays

Compound 1 blocks FcεRI-dependent activation of Cord-Blood DerivedPrimary Human Mast Cells (CHMC) in a dose-dependent manner with an EC₅₀of approximately 43 nM as assessed by measuring the activity of tryptasereleased upon degranulation. Compound 1 does not inhibitionomycin-induced degranulation of CHMCs. Ionomycin is a calciumionophore that induces CHMC degranulation bypassing early FcR signaling,thus indicating that Compound 1 is specific to FcR signaling, and notdegranulation per se. Compound 1 also inhibits the FcεRI-dependentproduction and release of LTC4 (EC₅₀=39 nM) and all cytokines tested(EC₅₀ ranging from 158 nM-462 nM).

12. Drug Compound 1 is Effective in Animal Models of RheumatoidArthritis

The biologic activity of Compound 1 in IC-mediated vascular edema(Arthus reaction in the rat), in collagen antibody-induced arthritis inthe mouse, and in a rat model of collagen-induced arthritis.

12.1. Arthus Reaction

IC-mediated acute inflammatory tissue injury is implicated in a varietyof human autoimmune diseases, including vasculitis, serum sickness,systemic lupus erythematosus, RA, and glomerulonephritis. The classicalexperimental model for IC-mediated tissue injury is the Reverse PassiveArthus (RPA) reaction. Intravenous injection of antigen (ovalbumin, OVA)following intradermal injection of antibodies specific to OVA (rabbitanti-OVA IgG) results in perivascular deposition of IC and a rapidinflammatory response characterized by edema, neutrophil infiltration,and hemorrhage at the injection sites (Szalai, et al., 2000, J. Immunol.164(1):463-468).

A single oral treatment of rats with Compound 1 one hour prior toantigen/antibody administration reduced the cutaneous RPA reaction andinflammatory edema in a dose-dependent manner. Administration of 10mg/kg oral Compound 1 inhibited extravascular leakage of Evans blue dye(OD₆₁₀) from tissue biopsies by 80% compared with vehicle control.

12.2. Collagen Antibody-Induced Arthritis

The anti-inflammatory activity of Compound 1 was evaluated in the mousecollagen antibody-induced arthritis (CAIA) model in which an anti-typeII collagen antibody cocktail is applied to induce arthritis (Teroto etal., 1992, J. Immunol. 148(7):2103-2108; McCoy et al., 2002, J. Clin.Invest. 110(5):651-658; Kagari et al., 2002, J. Immunol.169(3):1459-1466). This passive model differs from the traditionalrodent collagen-induced arthritis (CIA) in that disease symptoms appearquickly (developing within 24-48 hrs after an IV-injection ofantibodies), arthritis is inducible in both CIA-susceptible andCIA-resistant mouse strains, and it allows evaluation of inflammationthat is independent of antibody production.

CAIA was induced in Balb/c mice by intravenous injection ofArthrogen-CIA® Monoclonal Antibody Blend (Chemicon International, Inc.,Temecula, Calif.) via the tail vein, followed 2 days later by anintraperitoneal injection of LPS. Oral Compound 1 treatment was startedwithin 4 hours of antibody administration (Day 0). The severity of thearthritis in hind-paws was scored daily (scale of 0-4 per paw, sum ofscores for both hind paws). By Day 5, both control groups, saline andvehicle, reached their peak clinical score with a disease incidence of100%.

Reduced inflammation and swelling was evident in animals treated withCompound 1, and the arthritis progressed more slowly. Treatment withCompound 1 (b.i.d.) significantly reduced clinical arthritis (p<0.05)compared with animals treated with vehicle only, while lower dose levelsof Compound 1 showed a trend toward reduced arthritis severity, diseaseincidence, and time of onset; however, the differences were notsignificant (p>0.05).

12.3. Collagen-Induced Arthritis

One of the experimental models for IC-mediated tissue injury is the CIAin rodents (Kleinau et al., 2000, J. Exp. Med. 191:1611-1616). Injectionof type II collagen (CII) into rodents produces an immune reaction thatcharacteristically involves inflammatory destruction of cartilage andbone of the distal joints with concomitant swelling of surroundingtissues. CIA in rats is commonly used to evaluate compounds that mightbe of potential use as drugs for treatment of rheumatoid arthritis andother chronic inflammatory conditions and is induced in susceptiblestrains of either mice or rats by injection of CII in incompleteFreund's adjuvant (IFA). Administration of this emulsion gives rise topolyarthritis, characterized by synovial hyperplasia, infiltration ofmononuclear cells, pannus formation, and destruction of cartilage andbone. It has been previously well documented that antibodies to CII area prerequisite for CIA in mice, as B-cell deficient mice do not developarthritis (Svensson et al., 1998, Clin. Exp. Immunol. 111:521-526).

Syngeneic LOU rats were immunized on Day 0 with native chicken CII/IFA.Oral treatment began at the onset of arthritis symptoms (Day 10). Atotal of 59 rats were treated with either a vehicle control or Compound1 at one of four dose levels (1, 3, 10, and 30 mg/kg, q.d. by p.o.gavage). Hind limbs were scored daily for clinical arthritis severityusing a standardized method based on the degree of joint inflammation.High resolution digital radiographs of hind limbs were obtained at theconclusion of the study (Day 28). These limbs were also analyzed forhistopathologic changes. IgG antibodies to native CII were measured inquadruplicate by ELISA. There was a significant reduction (p<0.05) inarthritis severity that was evident within 7 days after initiation oftherapy in the high-dose (30 mg/kg) group that continued to improvethroughout the study. By Day 28, the clinical score in the animalstreated with vehicle alone was 6.08±0.67 compared to 2.54±0.98 in theCompound 1 30 mg/kg group (p<0.001). Blinded radiographs at studytermination (Day 28), demonstrated a significant reduction in jointdamage: 3.66±0.71 (vehicle) vs. 1.63±0.67 (Compound 1) (p<0.02) (E.Brahn. 2004). Blinded composite histopathologic studies confirmed theregression of pannus and erosions: Mean modified Mankin scores were11.8±0.9 (vehicle) vs. 3.7±0.9 (30 mg/kg Compound 1) (p<0.001).Antibodies to native CII were not decreased in Compound 1-treated rats.

13. The Prodrug Compounds Are Orally Bioavailable

Prodrug Compound 4 was tested for oral bioavailability. For the study,the prodrug was dissolved in various vehicles (e.g. PEG 400 solution andCMC suspension) for intravenous and oral dosing in the rats. Whereindicated, the active metabolite Compound 1 compound (drug) wasformulated and administered in the same vehicles. Followingadministration of the prodrug and/or drug, plasma samples were obtainedand extracted. The plasma concentrations of the prodrug and/or drug weredetermined by high performance liquid chromatography/tandem massspectrometry (LC/MS/MS) methods. Pharmacokinetic analyses were performedbased on the plasma concentration data. The pharmacokinetic parametersof interest include Clearance (CL), Volume of distribution atsteady-state (Vss), terminal half-life (t ½), and oral bioavailability(% F).

The results of these various pharmacokinetic experiments are illustratedin FIGS. 4-12.

Referring to FIG. 4, PK profiles are shown for IV and PO administrationin Sprague-Dawley rats. For IV administration, Compound 4 was dissolvedin PEG-400 and administered at a dose of 1 mg/kg. Rapid disappearance ofprodrug Compound 4 was observed and drug Compound 1 was found in plasmasamples obtained from the jugular vein. Given orally in the samevehicle, no prodrug Compound 4 was present systemically, but high levelsof drug metabolite Compound 1 were observed.

FIG. 5 summarizes the PK parameters for the study described in FIG. 4.Prodrug Compound 4 is rapidly cleared and, in part, converted to drugCompound 1. Given orally at a dose of 4 mg/kg, bioavailability wasdetermined to be 29.9%. This bioavailability number is based on dataobtained from a previous study (data not shown) in which drug Compound 1was administered as an IV bolus dose at 1 mg/kg.

FIG. 6 compares drug Compound 1 exposure in Sprague-Dawley ratsfollowing oral administration of either drug Compound 1 (2.5 mg/kg inPEG-400) or prodrug Compound 4 (4 mg/kg in PEG-400). The values forAUC/dose are nearly identical indicating that the prodrug Compound 4 isabsorbed equally as well as drug Compound 1.

FIG. 7 shows a plot of cLogD vs pH calculated using in-situ predictionsfor both Compound 1 and Compound 4. Compound 1 is highly liphophyllicand only weakly ionizable (measured solubility is less than 1 mcg/ml inphosphate buffer at pH=7.5, data not shown). In contrast, Compound 4 ishighly polar at neutral pH. Measured solubility values are consistentwith the predicted cLogD values at pH=7.5.

FIG. 8 demonstrates that prodrug Compound 4 is stable under acidic andneutral conditions at 37° C.

FIG. 9 illustrates the conversion of prodrug Compound 4 to drug Compound1 in microsome preparations. Prodrug Compound 4 failed to convert todrug Compound 1 in microsomal preparations obtained from Xenotech. Infollow-up studies using intestinal and hepatic microsomes obtained froma different source, conversion of Compound 4 to Compound 1 was observed(data not shown).

FIG. 10 illustrates that prodrug Compound 4 is unstable in ratplasma—hydrolysis to drug Compound 1 is observed and the conversion toCompound 1 is thought to be catalyzed by phosphatase enzymes. Thepresence of Phosphatase activity in rat plasma was confirmed usingp-nitrophenyl phosphate—a known substrate for phosphatase.

FIG. 11 illustrates the absorption of prodrug Compound 4 from differentvehicles. Unlike drug Compound 1, absorption of prodrug Compound 4 isnot dependent on formulation. Prodrug Compound 4 is absorbed equallywell in solution formulations (PEG-400 and carboxymethylcellulose (CMC))and as a powder in hard gelatin capsules.

Based on the pharmacokinetic data, the oral bioavailability (% F) ofprodrug Compound 4 from all three vehicles tested (PEG-400 solution; CMCSolution; and powder in capsules) was determined to be approx. 30%.

14. Prodrug Salt Hydrates are Stable

Prodrug salt hydrate 32 was subjected to thermal analysis, moisturesorption analysis, and X-ray powder diffraction (XRPD) analysis todetermine its stability and crystallinity.

Differential scanning calorimetry (DSC) thermograms were obtained usinga DSC Q 100 (TA Instruments, New Castle, Del.). The temperature axis andcell constant of the DSC cell were calibrated with indium (10 mg, 99.9%pure, melting point 156.6° C., heat of fusion 28.4 J/g). Samples(2.0-5.0 mg) were weighed in aluminum pans on an analytical balance.Aluminum pans without lids were used for the analysis. The samples wereequilibrated at 25° C. and heated to 250-300° C. at a heating rate of10° C./min under continuous nitrogen flow. Thermogravimetric analysis(TGA) of the samples was performed with a Q 50(TA Instruments, NewCastle, Del.). Samples (2.0-5.0 mg) were analyzed in open aluminum pansunder a nitrogen flow (50 mL/min) at 25° C. to 210° C. with a heatingrate of 10° C./min.

The sample for moisture analysis was allowed to dry at 25° C. for up to4 hours under a stream of dry nitrogen. The relative humidity was thenincreased stepwise from 10 to 90% relative humidity (adsorption scan)allowing the sample to equilibrate for a maximum of four hours beforeweighing and moving on to the next step. The desorption scan wasmeasured from 85 to 0% relative humidity with the same equilibrationtime. The sample was then dried at 80° C. for 2 hours or until no weightloss was observed under a stream of dry nitrogen.

X-ray powder diffraction data were collected using a Miniflex TabletopXRD system (Rigaku/MSC, The Woodlands, Tex.) from 5° to 45° 2θ withsteps of 0.1°, and the measuring time was 1.0 second/step. All sampleswere ground to similar size before exposure to radiation. The powdersamples were illuminated using CuKα radiation (λ=1.54056 Å) at 30 kV and15 mA.

Variable temperature XRPD data were collected using a Huber ImagingPlate Guinier Camera 670 employing Ni-filtered CuKα₁ radiation(λ=1.5405981 Å) produced at 40 kV and 20 mA by a Philips PW1120/00generator fitted with a Huber long fine-focus tube PW2273/20 and a HuberGuinier Monochromator Series 611/15. The original powder was packed intoa Lindemann capillary (Hilgenberg, Germany) with an internal diameter of1 mm and a wall thickness of 0.01 mm. The sample was heated at anaverage rate of 5 Kmin⁻¹ using a Huber High Temperature Controller HTC9634 unit with the capillary rotation device 670.2. The temperature washeld constant at selected intervals for 10 min while the sample wasexposed to X-rays and multiple scans were recorded. A 2θ-range of4.00-100.0° was used with a step size of 0.005° 2θ.

FIG. 12 presents the DSC and TGA thermograms of a sample of prodrug salthydrate 32. The DSC thermogram reveals three endothermic transitions: aninitial peak with an onset temperature of 42.98±2.01° C. and a peaktemperature of 70.26±1.78° C., immediately followed by another endothermwith an onset of 90.33±3.21° C. and a peak at 106.19±2.89° C. Theenthalpy value associated with the first transition is 209.70±12.34 J/gwhile the second transition requires about 67.24±4.56 J/g. In accordancewith the DSC transitions, TGA analysis indicates three distinct stagesof weight change. During the first step, the sample loses 9.64±0.18% ofmass followed by a subtle change in slope leading to another weight lossof 5.13±0.11%. The sample thus loses a total of about 14.74±0.14% up toa temperature of 150° C. These results are in harmony with Karl Fishertitration data which indicates that the water content of the sample isapproximately 14.82±0.17%, corresponding to 6 moles of water per mole ofprodrug salt. Thus, the first two transitions in the thermal profile ofprodrug salt 32 are due to dehydration of the sample. The final DSCtransition starts at 206.54±4.63° C. with peak maxima at 223.35±2.27° C.and enthalpy of 35.26±5.44 J/g and is thought to be due to melting. TheTGA indicates a weight loss during this transition, which is attributedto mass loss due to decomposition of the sample.

The moisture sorption profile of a sample of prodrug salt hydrate 32,shown in FIG. 13, demonstrates that the hexahydrate form is stable overa wide range of relative humidities. When the sample is dried at ambienttemperature and about 0% relative humidity the sample tends to losemajority of its moisture content. As observed in the FIG. 13, themoisture content of the sample is approximately 4.0% under dryconditions. This indicates that approximately 4 water molecules perprodrug salt 32 are lost at this low humidity. However, as soon as thehumidity is increased, the sample regains all of its water molecules andreturns to approximately 14.0% water content. Notably, this shift from4.0 to 14.0% water occurs in a very narrow range (0-10%) of relativehumidities. Compared to the change occurring in this narrow humidityrange, the change in moisture content over 10.0 to 95.0% relativehumidity is relatively small.

While not intending to be bound to any particular explanation, theinventors surmise that this behavior can be explained by the behavior ofan isomorphous solvate-desolvate system. At low humidity, the sampleloses majority of the water molecules, yet retains the three dimensionalorder of the crystal lattice. The dehydrated, unrearranged latticeremains extremely hygroscopic, and resorbs water as soon as the humidityis increased. Once the water molecules have been regained, the latticebecomes stable, and no further change in hydration is observed.

FIG. 14 presents the XRPD pattern of a sample of prodrug salt 32. As isevident from the pattern, the sample has a well defined crystalstructure and a high degree of crystallinity. The XRPD data issummarized in Tables 1 and 2, below.

TABLE 1 Peak Search Report (39 Peaks, Max P/N = 37.3) X-ray SourceSource Cu Filter Ni kV 30 mA 15 Kα□□  1.54059 (□Å)□□ scan  3 to 45 scan 2.00/min speed Method continuous Count cps

TABLE 2 XRPD pattern of prodrug salt 32 (PEAK: 47-pts/Parabolic Filter,Threshold = 3.0, Cutoff = 0.1%, BG = 3/1.0, Peak-Top = Summit) 2-Thetad(Å) Height H % FWHM XS(Å) 3.380 26.1226 3315 56.7 0.198 3488 6.62013.3412 1468 25.1 0.225 982 9.890 8.9360 1547 26.5 0.218 953 11.7507.5252 1192 20.4 0.292 443 12.350 7.1610 813 13.9 0.268 517 13.1506.7273 5846 100.0 0.362 305 13.510 6.5489 823 14.1 0.421 244 15.0205.8938 423 7.2 0.343 326 15.710 5.6365 186 3.2 0.163 3043 16.421 5.3940997 17.0 0.253 545 17.190 5.1543 1696 29.0 0.257 518 17.661 5.0177 1131.9 0.110 >5000 18.060 4.9079 206 3.5 0.216 743 18.600 4.7667 849 14.50.225 665 19.730 4.4960 2948 50.4 0.277 440 21.240 4.1797 1745 29.80.419 237 21.750 4.0828 1479 25.3 0.308 362 22.479 3.9521 100 1.70.138 >5000 23.400 3.7985 458 7.8 0.323 334 24.237 3.6692 123 2.1 0.240533 25.280 3.5201 1053 18.0 0.613 148 25.840 3.4451 1502 25.7 0.723 12327.150 3.2818 1037 17.7 0.508 183 27.510 3.2397 1563 26.7 0.477 19828.640 3.1143 644 11.0 0.269 423 29.470 3.0285 931 15.9 0.331 313 30.5312.9256 308 5.3 0.262 436 31.640 2.8256 1761 30.1 0.412 235 33.650 2.6613549 9.4 0.472 199 34.110 2.6264 670 11.5 0.603 150 34.990 2.5623 140 2.40.051 >5000 36.379 2.4676 248 4.2 0.467 202 37.251 2.4119 494 8.5 0.393248 38.540 2.3341 337 5.8 0.411 235 39.030 2.3059 519 8.9 0.582 15739.631 2.2723 172 2.9 0.210 576 40.650 2.2177 306 5.2 0.198 634 41.6402.1672 767 13.1 0.517 180 43.482 2.0796 155 2.6 0.531 175

Variable temperature XRPD data, shown in FIG. 15, provides furtherevidence of the existence of an isomorphous solvate-desolvate system. Asis evident in FIG. 15, the powder patterns obtained at 25° C. and 60° C.are substantially identical. From the thermal analysis described above,it is known that the sample starts dehydrating as early as 40° C. andpeaks at around 70° C. to lose 4 of its 6 water molecules. The XRPD doesnot indicate any structural changes at 60° C., suggesting that the lossof water molecules does not change the three dimensional order of thecrystal lattice. Structural changes in the sample are noted as thetemperature rises to about 110° C. As the thermal data demonstrates,almost all of the water molecules are lost around this temperature. Thissuggests that once the all of the water molecules are lost, the crystaldoes undergo structural rearrangement. As suggested by the loss of peaksin the XRPD, as the temperature increases the crystal lattice collapses,and the material eventually decomposes.

Single crystal x-ray diffraction of prodrug salt hydrate 32 wasperformed, and indicated that the crystal of the invention containssubstantially no methanol, and six molecules of water per prodrugmolecule. The experimentally determined crystal unit cell parameters anddata collection parameters for prodrug salt hydrate 32 are summarized inTable 3.

TABLE 3 Single crystal x-ray diffraction data for prodrug salt hydrate32. Crystal Data Formula C23 H36 F N6 Na2 O15 P Formula Weight  732.53Crystal System Monoclinic Space Group P2_(1/C) (No. 14) a (Å)  27.7282(5) b (Å)   7.9776(1) c (Å)   15.1739(3) alpha (°)   90 beta(°)  103.206(1) gamma (°)   90 V (Å³)  3267.8(1) Z   4 D (Calc) [g/cm³]  1.485 μ (MoKalpha) [/mm]   0.194 F(000)  1528 Crystal size [mm] 0.80 ×0.15 × 0.02 Data collection Temperature (K)  113 Radiation (Å) MoKalpha0.71073 Theta Min-Maz [Deg}   3.4, 27.8 Dataset −36:36; −10:10; −19:19Tot., Uniq data, R (int.) 26192, 7582, 0.089 Obsxerved Data [I > 2.0sigma(I)]  4452 Refinement Nref, Npar  7582, 474 R, wR2, S   0.0514,0.1312, 1.06 w = 1/[\s{circumflex over ( )}2{circumflex over( )}(Fo{circumflex over ( )}2{circumflex over ( )}) +(0.0587P){circumflex over ( )}2{circumflex over ( )} + 0.9541P] where P= (Fo{circumflex over ( )}2{circumflex over ( )} + 2Fc{circumflex over( )}2{circumflex over ( )})/3 Max. and Av. Shift/Error   <0.001, <0.001Min. and Max. Resd. Dens. [e/Å³]   −0.52, 1.27

The behavior of prodrug salt hydrate 32 indicates that it can beexpected to have long-term stability upon storage. The hexahydrate formis stable over a wide range of relative humidities, and requiressubstantial heating (e.g. greater than 40° C.) to begin to lose includedwater molecules. High humidity does not affect the hexahydrate;extremely low humidity (i.e. less than 10% RH) can cause dehydration ofthe hexahydrate, but re-exposure to ambient conditions engendersreformation of the hexahydrate. The lack of observed structural changesunder low humidity conditions, and heating up to 110° C., allows forwater loss to be reversible the loss to be reversible upon cooling.Additionally, the prodrug salt hydrate 32 has increased solubility (10mg/mL in water) with respect to the parent phosphate prodrug 4 and thecalcium salt, prodrug salt 6 (15 μg/mL in water).

The invention claimed is:
 1. An oral dosage formulation comprising a hydrate of the prodrug salt:

or a pharmaceutically-acceptable carrier, excipient, and/or diluent; wherein: M is Na; the prodrug salt hydrate present in the oral dosage formulation is substantially crystalline prodrug salt hydrate; and the powder X-ray diffraction data of the crystalline prodrug salt hydrate comprises a characteristic peak at a two theta value of 9.9±0.1° in response to radiation of wavelength 1.54059 Å.
 2. The oral dosage formulation of claim 1, the formulation comprising from about 50 mg to about 400 mg of the crystalline prodrug salt hydrate.
 3. The oral dosage formulation of claim 1, wherein the powder X-ray diffraction data of the crystalline prodrug salt hydrate comprises a characteristic peak at a two theta value of 11.8±0.1°.
 4. The oral dosage formulation of claim 1, wherein the powder X-ray diffraction data of the crystalline prodrug salt hydrate comprises a characteristic peak at a two theta value of 21.8±0.1°.
 5. The oral dosage formulation of claim 1, wherein the powder X-ray diffraction data of the crystalline prodrug salt hydrate comprises a characteristic peak at a two theta value of 13.2°±0.1°.
 6. The oral dosage formulation of claim 1, wherein the hydrate of the prodrug salt has the formula:

wherein x is from about 1 to about
 15. 7. The oral dosage formulation of claim 6, wherein x is from about 5 to about
 10. 8. The oral dosage formulation of claim 6, wherein x is from about 5 to about
 8. 9. The oral dosage formulation of claim 6, wherein x is
 6. 10. The oral dosage formulation of claim 8, wherein the powder X-ray diffraction data of the crystalline prodrug salt hydrate comprises a characteristic peak at a two theta value of 3.4°±0.1°.
 11. The oral dosage formulation of claim 1, wherein the hydrate is: 